Masters Degrees (Microbial, Biochemical and Food Biotechnology)

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Open Access
Ondersoekinge oor die afbraak van aromatiese verbindings deur mikro-organismes
(University of the Free State, 1960-01) Scott, De B.; Lütjeharms, W. J.
English: 1. In a survey of the relevant literature the methods of investigation, the systematic and biochemical aspects and the significance of microbial degradation of aromatic compounds has been discussed. 2. In a preliminary investigation it was found that nonselectivily isolated strains of streptomycetes dit not grow on a mineral salt medium with a simple aromatic compound as sole source of carbon. 3. Bacteria, fungi and one Nocardia strain able to use aromatic compounds as sole source of carbon and energy, were isolated from enrichment cultures. 4. From turbidimetric growth measurements it was found that optimum cell development of the bacteria and actinomycete investigated in a medium with a low concentration of aromatic substrate in most cases took place within two days. 5. The oxidation of a number of aromatic compounds by the bacteria and actinomycete isolated, was studied with the aid of the direct method of Warburg. 6. The method used for the calibration of manometers and Warburg flasks is described. 7. The technique of sequential induction was applied to determine the pathway of microbial degradation of the aromatic compounds investigated. With this technique the following hitherto unpublished reactions of bacteria have been found: (i) Salicylic acid - gentisic acid. (ii) Saligenin - salicylic aldehyde - salicylic acid - catechol and gentisic acid. (iii) Syringic acid - gallic acid. (iv) Guaiacol - catechol. (v) Vanillin - vanillic acid - protocatechuic acid. (vi) Veratric aldehyde - veratric acid - vanillic acid - protocatechuic acid. The degradation of phenol, benzoic acid, mandelic acid, p-cresol, m-hydroxybenzoic acid and anisic acid by bacteria, and of toluene by Nocardia sp., as studied with manometrical methods, was found to be identical with the pathway already described for bacteria and fungi. The degradation pathway of o- and m-cresol could not be established with certainty with the method used.
Open Access
Seisoenvariasie in die aminosuursamestelling van rumenprotosoë
(University of the Free State, 1978-02) Cilliers, Johannes Jacobus le Roux; Du Toit, P. J.
English: The rumen content of sheep was fractionated and analyzed. Samples were obtained over a year, at three week intervals, from a group of six Merino sheep on natural veld. The rumen contents of another group receiving specified crude protein feed and kept indoors under controlled conditions, were also analyzed. Rumen samples were fractionated into protozoal-, bacterial-, fibrous- and soluble fractions by a procedure of centrifugation. Sampling and conditions under which the sheep were kept as well as fractionating procedures are described and discussed. The rumen samples as well as the different fractions were analyzed by various means to determine the effects of seasonal variation. Dry mass, nitrogen (micro-Kjeldahl technique) and amino acid composition of these fractions were determined. In some cases the total carbohydrate- and tryptophane contents were determined. Observation of the different fractions by microscope was used for comparative purposes. For analytical determinations, including hydrolysis procedures, all sampLes were well mixed, homogenized where necessary, freeze-dried and pulverised to facilitate representative sampling. The amount of sample needed for amino acid determination was calculated from the nitrogen content. Samples were hydrolized for 22 hours at 110 °C in constant boiling HCl under vacuum. Hydrochloric acid was removed by evaporation and freeze-drying after hydrolysis and the amino acids transferred quantitavely into buffer for analysis. Amino acid analysis were conducted on a Beckman Model 120 C amino acid analyzer according to a standardized procedure. The analysis were controlled for accuracy and values compensated for losses of certain amino acids caused by acid hydrolysis. According to results, the nitrogen as well as the total amino acid content varied with seasonal variations with minimum values at the end of the winter. Each amino acid, viewed as a percentage of the total amino acids in the fraction, remained constant within certain limits over the full duration of the experiment. Certain essential amino acids, i.e. Lys, lIe and Arg occured at comparatively higher concentration levels in the protozoal fraction, whereas Thr, Ala, Tyr and Met were present at higher levels in the bacterial fraction as compared to the rumen sample. Pro and His seem to be degraded to a marked extent in the rumen, whereas Asp and Glu occured at relatively high concentration levels in all fractions, including the feed. The quality and composition of fodder have a marked effect upon the microflora of the rumen. The sheep on crude protein feed displayed much higher levels of protozoa (dry mass) than bacteria. The opposite pattern was observed with sheep on the veld. Protozoa from sheep on crude protein feed also had higher concentration levels of Lys, Tyr, Arg, Asp and Glu but lower ,levels of Ala and Gly. Bacterial fractions from both groups showed very little difference in amino acid composition. These results seem to indicate a definite role for protozoa in the rumen especially when the animal is fed a high quality fodder. In addition to preferentially supplying certain essential amino acids (protozoa used as feed) these organisms also seem to aid in the break-down of complex macromolecules present in the feed. In this respect a glycoside hydrolase, obtained from the protozoal fraction of grazing sheep, was partially purified and studied. The enzyme was purified by ethanol fractionation as well as chromatographic procedures. The optimum pH as well as the activity towards various carbohydrates was also determined. The occurence of specific hydrolases in the different micro-organisms is also affected by the feed supplied to the animal. It seems essential that further studies specifically concerning the metabolic role of protozoa in the rumen should be done.
Open Access
Long-chain fatty acid compositions and volatile metabolite patterns of yeasts associated with wine
(University of the Free State, 1987-05) Tredoux, Hendrik Gabriël; Kock, J. L. F.
A) In Chapter 1 the need for a yeast identification system in the wine industry is highlighted. The definition, as well as taxonomic development of the ascomycetous yeasts are discussed as well as the problems encountered. B) In Chapter 2 the cellular long-chain fatty acid compositions of 103 yeast strains representing 38 species related to the wine industry were determined gaschromatographically. It was possible to differentiate between most species examined as well as between some strains within species. A correlation was observed between long-chain fatty acid composition and complexity of cell differentiation, genetic recombination, carbon source- and ethylamine utilization and resistance to cycloheximide. A phylogenetic scheme for the genus Kluyveromyces was constructed on the basis of the abovementioned features. C) Chapter 3 includes the use of volatile metabolites in the identification of winery-associated yeasts. According to the results it was possible to differentiate between the Saceh. cerevisiae and S. pombe strains. D) A Discussion and Conclusions is presented in Chapter 4. This includes a discussion on the identification of wine yeasts and the relation between long-chain fatty acid composition, pseudomycelium formation, genetic recombination, carbon source- and ethylamine utilization and resistance to cycloheximide. A possible relation between the similarity in long-chain fatty acid compositions and DNA homology between yeasts strains is indicated. The use of volatile metabolites in the identification of wine yeasts is also discussed.
Open Access
Design, synthesis and expression in different hosts of a gene coding for a small multifunctional peptide
(University of the Free State, 1994-01) Van der Merwe, Walda Brenda; Pretorius, G. H. J.; Kotzé, H. F.
Platelets and coagulation both play a pivotal role in thrombosis, one of the major causes of death in Western society. It is therefore not surprising that potent inhibitors are being developed to inhibit platelet function or coagulation. In this study we developed a multifunctional peptide that would not only inhibit thrombin, but will also prevent platelet aggregation. A 29 amino acid peptide was designed comprising three inhibitory regions: 1, a part of hirudin, a potent direct antithrombin; 2, fibrinopeptide A, also an inhibitor of thrombin and 3, an amino acid sequence (arqgly- asp) essential for binding to the fibrinogen receptor on platelets to prevent fibrinogen binding. The protein sequence was reverse translated and two 60-mers with an overlap of 22 base pairs were synthesized. After filling in the ends, the gene was cloned into a yeast expression and secretion vector, pMFα8. The construct was sequenced to verify correct orientation of the gene and transformed into yeast. The expected peptide of approximately 3 kDa could not be seen on SDS-PAGE gels, nor could inhibition of thrombin or platelet aggregation be shown. However, Northern hybridization analysis revealed the presence of the mRNA. The introduction of a methionine residue at the N-terminus of the peptide and the use of protease-deficient yeast strains as expression hosts, did not improve peptide production. Another expression system, the inducible yeast expression vector pYES2, was then employed. Although no peptide could be detected, Northern blotting showed the presence of high levels of the mRNA. Optimal codons for expression in yeast were selected in the design of the gene, but the absence of a detectable level of peptide could be due to a low translation rate or due to proteolysis. Alternatively, the gene was cloned into a regulatable E. coli vector, pQE-32, and expressed intracellularly in E. coli. No band of the correct size could be detected on SDS-PAGE gels, which might be due to the extremely small molecular size of the peptide.
Open Access
The isolation of gamma-linolenic acid producing mucoralean fungi
(University of the Free State, 1997-11) Strauss, Tersia; Botha, A.; Kock, J. L. F.
English: Members of Mucorales are known to produce the high value fatty acid gamma-linolenic acid [18:3(ω6)]. Although few studies have been conducted, it is known that the type of carbon source included in the medium, influences the production of 18:3(ω6) by these fungi. The range of carbon sources on which mucoralean fungi are able to grow and produce 18:3(ω6), is still mostly unknown. Another factor that influences the quantities of 18:3(ω6) that are being produced by these fungi, is the specific fungal strain that is used in the process. Consequently, in this study it was decided first to investigate the ability of different mucoralean fungi to grow and produce 18:3(ω6) on a wide range of carbon sources. Isolation media for obtaining new strains from nature, which utilize carbon sources obtainable from industrial effluents, would subsequently be developed. The influence of 38 different carbon sources on growth and consequent 18:3(ω6) content of the lipids produced by four mucoralean fungal strains were therefore investigated. The strains represented the species Morlierella afpina, Mucor circinelloides, Mucor ffavus and Thamnosfyfum piriforme. The representatives of M. circinelloides and M. ffavus respectively utilized 25 and 23 of the 38 carbon sources in the series. The highest percentages 18:3(ω6) obtained with the representatives of M. circinelloides and M. ffavus were 27.17 % and 36.40 % respectively. In contrast, the highest percentages 18:3(ω6) obtained with the representatives of Mo. afpina and T. piriforme were only 5.61 % and 12.84 % respectively. These two strains could respectively utilize only seven and 17 of the carbon sources. This study indicated that mucoralean fungi can grow and produce 18:3(ω6) on a variety of carbon sources, including carbon sources present in industrial effluents (e.g. starch, sucrose and acetic acid). Three selective media were subsequently developed in order to isolate mucoralean fungi from soil, using the soil plate technique. The media, which were complex, respectively contained starch, sucrose and sodium acetate as carbon sources, as well as 0.02 g/l of the anti-fungal agent, benlate. The selectivity of the media for members of Mucorales was first determined by testing the media for the ability to support growth of 134 mucoralean fungal strains representing 66 species and seven genera. The three isolation media supported growth of strains representing Absidia, Actinomucor, Backusella, Mucor, Rhizopus and Thamnostylum. The ability of the isolation media to select mucoralean fungi from a natural fungal population in soil, was then determined and representatives of the genera Absidia, Cunningham'ella, GongronelIa, Mucor and Rhizopus were obtained. The results further showed that by using selective media in combination with a relatively non-selective medium, instead of the non-selective medium alone, more mucoralean taxa could be isolated from a particular soil sample. Mucoralean fungal isolates that were obtained from the soil sample, were subsequently evaluated for growth and 18:3(ω6) production in media containing starch, sucrose or glucose as sole carbon sources. Isolates representing the families Absidiaceae, Cunninghamellaceae and Mucoraceae were inoculated in complex media containing the above mentioned carbon sources. It was found that all the isolates were able to produce 18:3(ω6) on all three carbon sources. However, significant differences in volumetric 18:3(ω6) concentrations reached on different carbon sources were noted for each isolate investigated. The highest volumetric concentrations of 18:3(ω6) were obtained with an isolate representing R. stolonifer on starch (0.130 gii) and glucose (0.134 gii) as carbon sources. In order to prove that the isolates obtained using the above-mentioned isolation media, are able to grow in an industrial effluent, some of the isolates representing different families, were grown in a medium prepared from an industrial effluent containing dextrins, galactans and starch as carbon sources. The lipids of the isolates which reduced the COD value of the effluent the most, were analysed. It was found that these isolates were able to produce 18:3(ω6). This study has therefore shown that it is possible to construct isolation media to isolate 18:3(ω6) producing mucoralean fungi from a natural fungal population. It was also found that such isolates can be used to produce biomass and 18:3(ω6) from carbon sources present in industrial effluents.
Open Access
Cryopreservation and chemotaxonomy in Saccharomyces meyen ex reess
(University of the Free State, 1998-11) Morakile, Gontse; Kock, J. L. F.
English: The ability to preserve microorganisms can be considered a major . biological achievement. Of special importance is the understanding of the principles of culture preservation with minimal occurrence of contamination, genetic and viability change. At present, cryopreservation is considered the most successful preservation method for yeasts yielding high survival levels and good phenotypic stability. As a result, one of the aims of this study was the application and evaluation of a cryopreservation protocol used in the maintenance of a Saccharomyces cerevisiae strain used by a major brewing company in South Africa. In order to ensure that only pure and stable yeasts with high viability are used after revival from maintenance protocol, it is essential that appropriate, rapid and inexpensive quality control methods are implemented. Elaborate and time consuming tests are used today in the brewing industry and include estimation of mutants and bacteria using Wallerstein Laboratory Nutrient Medium (WLN), estimation of respiratory deficient (RDs) yeasts using wort agar overlaid with Triphenyl- Tetrazolium-Chloride, detection of the wild yeasts using the Swartz-Differential Medium (SDM) protocol, estimation of the non-Saccharomyces species using Lysine-Medium (LYS), detection of the lactose assimilating and lactose fermenting microorganisms using the Lactose-Peptone-Broth (LP) and detection of brewery bacteria using the Universal Liquid Medium (ULM). According to the results, a decrease in the percentage RDs as well as Variants (i.e. other mutants of Saccharomyces cerevisiae) was evident in brewing inocula when maintained through cryopreservation. When yeasts from slants (not cryopreserved) and yeasts subjected to cryopreservation were cultivated in wort contained in round bottom flasks, no significant changes in the percentage RDs, variants or maximum growth rate (J.lmax) could be detected. A 4x3x3x2 nested experimental design was performed in order . to determine the sources of variation in yeast viability, stability and contamination after preservation in liquid nitrogen for 136 days. Consequently, results on Variants and RDs suggest that the largest source of variation in the cryopreservation maintenance process was the error arising from the analytical tests. Cryopreservation also influenced the variation in the number of RDs obtained, though to a lesser extend. No contamination was found to occur during the cryopreservation protocol. From this study it is now possible to construct statistical quality control charts that can be used as an aid in the manufacture of brewing inocula maintained through cryopreservation. Another aim of this study was the evaluation of chemotaxonomie characters such as sterols and polar lipids in, as a first step, determining contamination of preserved yeasts with closely related species. According to the results, total lipid content as well as polar lipid fractions and associated fatty acid (FA) composition showed no obvious taxonomic value as the different Saccharomyces species could not be differentiated. Both linoleic acid (18:2) and linolenic acid (18:3) were detected in strains characterised by the absence of these FAs in total FA composition, a situation believed to be due to the 'dilution' of these FAs by the dominating NL fraction. Sterol content showed promise in the demarcation of the Saccharomyces sensu strictu group.
Open Access
The Amylostereum symbiont of Sirex noctilio in South Africa
(University of the Free State, 1998-12) Slippers, Bernard; Wingfield, M. J.; Coutinho, T. A.; Wingfield, B. D.
English: In Chapter 1 of this thesis, the literature pertaining to the symbiosis between Sirex noctilio and Amy/ostereum areo/atum in the Southern Hemisphere, is reviewed. It is evident from this review that S. noctilio and A. areo/atum have become established throughout the pine growing regions of the Southern Hemisphere, despite measures to prevent its introduction. Unlike its relative unimportance as a pathogen in the Northern Hemisphere, this fungal-insect complex has resulted in great losses to softwood industries during a number of severe outbreaks in the Southern Hemisphere. The use of biological control agents in combination with preventative silvicultural practices, has been shown to be very effective in controlling Sirex in Australasia. It is, however, also evident from this review that despite the rather large collection of knowledge concerning the wasp and its control, information regarding the population structure and phylogenetic relationships of the fungal symbiont of Sirex, is scarce. The recent introduction of S. noctilio into South Africa and its confinement to a rather small area in this country provided the opportunity to study the population of its fungal symbiont in detail. Results from Chapter 2 suggest that the fungus has a very narrow genetic base in South Africa and that the introduction of Sirex into this country was limited. The genetic base of A. areolatum in Brazil and Uruguay is similarly uniform. Of even greater interest is the fact that South Africa and Brazil share a common vegetative compatibility group and, thus, a common origin of A. areo/atum and S. noctilio. Moreover, field isolates from the Southern Hemisphere appear to be closely related, which indicates that Sirex might have spread among countries of the Southern Hemisphere and were not necessarily new introductions from the Northern Hemisphere. Isolates of the fungus associated with the biocontrol nematode, De/adenus siricidicola, are, however, distinct from isolates from other Southern Hemisphere populations of the fungus. This could negatively influence the efficacy of the nematode as biocontrol agent in countries to which the nematode has been distributed. Boidin and Lanquetin (1984) report triangular mating incompatibility between isolates from the different Amy/ostereum spp. Results of Chapter 3 support their conclusions by clearly showing that A. areolatum is more distantly related to A. chailletii, A. laevigatum and A. ferreum, than these three species are to each other. The relationship between the latter three species is, however, more clearly defined in Chapter 3 where it is shown that A. ferreum and A. laevigatum are most closely related to each other. One isolate collected from Sirex areolatus, and, therefore, expected to be A. chailletii, was most closely related to A. laevigatum and A. ferreum. Neither of the latter species has, however, been implicated in associations with woodwasps. Furthermore, the data from this study show that Amylostereum spp. group with neither Stereum nor Peniophora, as has been previously hypothesised, but rather with Echinodontium tinctorium. This grouping was included in a larger clade that included species of Russula, Heterobasidion, Lentinellus and Auriscalpium. Analysis of DNA sequence data derived from the nuc-IGS-rDNA in Chapter 4 supported the phylogenetic relationships of the Amylostereum spp. inferred in Chapter 3. Similarly, the isolate obtained from S. areolatus, did not group with any of the four species of Amylostereum and might represent a new species or a distinct group in of one of the current species. Isolates of A. areolatum associated with both S. noctilio and S. juvencus contained four heterogenic sequences in the DNA region analysed. These heterogenic sequences were contained in each isolate of the fungus in one of five combinations. Neither the heterogenic sequences included in the fungal isolates, nor the different combinations of these sequences, separated the populations of A. areolatum associated with different wasp species. Despite the heterogenic nature of this DNA region in some isolates, RFLP analysis was used effectively to distinguish between the different species of Amylostereum. The work presented in this thesis represents the first molecular. view of the phylogeny of the genus Amylostereum, as well as that of some of the Amylostereum spp. associated with woodwasp species. It is clear from Chapter 5 that these findings now provide a powerful tool to give a clearer picture of the taxonomy and evolution of these fungi, as well the ecology of their symbiosis with woodwasps. The study of the genetic structure of the fungal populations associated with woodwasps also gives new insight into the geographical origin and history of both the insects and their associated fungi.
Open Access
Oomycetes associated with citrus and eucalypts root rot in South Africa
(University of the Free State, 1999-03) Maseko, Bongani; Coutinho, Teresa; Wingfield, Michael J.; Wolfaardt, Francois
English: Research conducted in this thesis explores the role that Phytophthora and Pythium species play in citrus and eucalypt root rot in South Africa. The first chapter presents an extensive literature review with special emphasis on the importance of Phytophthora and Pythium spp. to the South African Citrus and Forestry Industries. This chapter is divided .into two parts. Part one deals with biotic and abiotic factors that contribute to citrus decline. Part two focuses on recently published work on root diseases of exotic forest trees species planted 'commercially in South Africa. Special emphasis was placed on P. cinnamomi since it is thought to be the most important pathogen in the forestry industry. Results obtained in Chapter Two indicate that a wide variation in tolerance and susceptibility to P. cinnamomi exits amongst half-sib families of E. fraxinoides. Field mortality of 52 % and 30% was recorded at two trial sites following natural infection. Seven disease tolerant families were identified with the potential to be used for commercial propagation. Eucalyptus fraxinoides families tolerant to P. cinnamomi were more reliably identified using stem inoculation of young trees relative to selecting families that survived the disease after planting in the .field. In Chapter Three half-sib families of E. fraxinoides and E. smithii were evaluated for tolerance to P. cinnamomi using stem inoculations in the greenhouse. Three replicate trials for each species were conducted at different times of the year. Disease tolerance and susceptibility varied between and among the different E. fraxinoides and E. smithii families. No correlation was found between the results obtained from the replicate trials of E. fraxinoides and E. smithii. The technique used was found to be unreliable for screening E. fraxinoides and E. smithii families for tolerance to P. cinnamomi. Results of a preliminary survey in selected citrus nurseries and orchards in the Northern and Mpumalanga provinces of South Africa are presented in Chapter Five. A total of 320 Phytophthora and Pythium isolates were recovered from the rhizosphere of diseased citrus trees and diseased plant material. Phytophthora nicotianae and Pythium irregulare where the most frequently isolated species. Sixteen isolates of P. citrophthora were successfully recovered. This species has previously been presumed to be absent from the sampled regions. In the last Chapter of this thesis, pathogenicity tests were conducted on Phytophthora and Pythium spp. associated with citrus root rot. Phytophthora isolates proved to be pathogenic when inoculated into citrus fruit while Pythium spp. where either weakly or non pathogenic. Contrary results were found when the lupin assay was conducted since all Pythium spp. proved pathogenic. Phytophthora isolates were also found to be pathogenic on Rough Lemon (RL) and Troyer Citrange (RTC) rootstocks whereas Pythium spp were either weakly pathogenic or avirulent. No difference in susceptibility was observed between RL and RTC. The fruit _assay and stem inoculation techniques were found to be reliable, inexpensive and rapid results were obtained. Results obtained in this study indicated that Phytophthora spp. play an important role in citrus and eucalypt root rot. Artificial stem inoculation using a single virulent isolate of P.cinnamomi proved to be a reliable technique for screening young eucalyptus trees in the field. However, contrary results were obtained when this technique was used to screen half-sib seedlings of E. fraxinoides and E. smithii in the greenhouse. Eight Pythium and two Phytophthora spp. were isolated from the rhizosphere soil and declining citrus trees. Phytophthora species proved to be pathogenic when inoculated into citrus fruit and on Rough Lemon and Troyer Citrange rootstocks. On the other hand Pythium species screened proved to be nonpathogenic or only weakly pathogenic and thus probably play an insignificant role in citrus decline.
Open Access
The hydrolysis of linalyl acetate and α-terpinyl acetate by yeasts
(University of the Free State, 1999-06) Thomas, Elias; Smit, M. S.; Litthauer, D.
English: Hydrolysis of esters by means of hydrolases such as proteases (Jones and Beck, 1976), lipases (Santiello et al., 1993) and esterases (Boland et al., 1991) has become a well established method for the resolution of racemic mixtures. The first aim of the present study was to screen the yeast culture collection of the University of the Orange Free State for yeast isolates which can be used for the enantioselective hydrolysis of rac-linalyl acetate and rac-α-terpinyl acetate, which are tertiary alcohol ester terpenes, respectively. We screened 74 yeast strains from 17 genera as well as 29 unclassified isolates. Approximately 16% of the strains screened contained tertiary alcohol hydrolase activity. Whole cell experiments, enzyme purification and characterisation were attempted on one of the hydrolases of interest obtained from Trichosporon sp. UOFS Y-0117. Whole cell experiments on reported optimal hydrolase activity in the presence of 1% maltose in a defined media (YNB). The effect of various co-solvents was also documented with a low concentration of ethanol (2.4% v/v) producing superior hydrolase activity. No toxicity, to the microbe, was observed by rac-linalyl acetate (up to 200mM) due to the cell membrane present. The use of digitonin proved that substrate transport across the cell membrane is not a reaction rate determining step. The re-usability experiment showed a significant decrease in hydrolase activity (ca 30% after 1 cycle) as the same batch of cells was exposed to substrate and product. The results also show an optimal pH of 7.5 and temperature of 30°C which coincides with physiological conditions and literature. Protein purification was attempted on a cell free extract once we determined that the hydrolase was intracellular. Small scale evaluations of different chromatographic resins ranging from ion exchange, hydrophobic interaction and affinity chromatography followed. Large scale experiments with gel filtration resins were also attempted. Purification steps were largely unsuccessful and we decided to continue with a DEAE fraction which produced a superior yield (244%). Characterisation experiments, using the DEAE active fraction, followed in which we explored the effect of rac-linalyl acetate concentrations. Enzyme inhibition and protein denaturation at low rac-linalyl acetate concentrations (detected at ca 65-100mM) is significant compared to whole cells (not detected at 200mM). This hydrolase is also an esterase. Specific amino acid modification reagents results indicate the presence of a serine and histidine amino acid present in the catalytic centre i.e. the hydrolase belongs to the serine hydrolase family. A metal chelating reagent EDTA and various metal cations had no effect on hydrolase activity. pH-stability experiments indicate a pH of 7.5 to be optimal for the retention of hydrolase activity in whole cells and crude enzyme preparation. Thermostability experiments show whole cells are four times more stable than the crude enzyme preparation at 4°C. The energy of inactivation required for activity loss is lower in whole cells (47.43kJ) compared to crude enzyme preparation (91.77kJ). The probability for an event to occur which causes inactivation is relatively low (1.8075 events/h). The converse applies to the crude- enzyme preparation (1.17514 events/h). Thus the crude enzyme is 6x108 less stable than the hydrolase present in the whole cell.
Open Access
Mucoralean fungi present in soil from arid regions in South Africa
(University of the Free State, 1999-11) Seabi, Buti Oscar; Botha, A.; Viljoen, B. C.
English: The aim of the first part of the study was to investigate the ecological niche of mucoralean fungi in arid soil, with specific reference to the position these fungi occupy in the biogeochemical cycle of nitrogen. Consequently, selected mucoralean taxa occurring frequently in soil habitats, including strains from culture collections, as well as isolates obtained from a soil sample from arid Upper Nama Karoo, were used to evaluate in vitro growth to determine nitrogen sources and aw tolerances. Nine mucoralean fungal genera including 18 species were examined for the ability to utilise a series of nitrogen containing compounds and to grow at an aw of 0.955 on solid media. The nitrogen concentration in the media was 0.1 g.r1 and the series of nitrogen containing compounds were ammonium chloride, asparagine, sodium glutamate, sodium nitrite and potassium nitrate. The genera were Actinomucor Schostak., Backusella Hesselt. & J.J. Ellis, Cunningha'fnella Matr., GongronelIa Ribaldi, MortierelIa Coem., Mucor Fresen., Rhizomucor Lucet & Costantin., Rhizopus Ehrenb. and Thamnostylum Arx & H. P. Upadhyay. Thirty-nine fungal strains obtained from culture collections (CBS, MUFS and PPRI), as well as 12 soil isolates from the Karoo, were tested. All the species and strains tested in this study were able to utilise asparagine and glutamate. Strains belonging to CunninghamelIa, Mucor racemosus Fresen., Rhizopus microsporus Tiegh. and Rhizopus stolonifer (Ehrenb.: Fr.) VuilI. were unable to utilise ammonium chloride. Strains of CunninghamelIa, MortierelIa, Rhizomucor, Rhizopus microsporus and Rhizopus stolonifer were unable to grow on nitrate as sole nitrogen source. Nitrite was found to be toxic to species belonging to CunninghamelIa, MortierelIa, Rhizomucor, Rhizopus and Thamnostylum. Members of GongronelIa, MortierelIa, Mucor racemosus, Rhizomucor and Thamnostylum were unable to grow at an a, of 0.955. The aim of the second part of the study was firstly to get an indication whether the mucoralean diversity of the Karoo, as observed in the first part of the study and in the records obtainable from literature, differs from data on mucoralean diversity from other arid regions. The latter included data from literature and what could be found in a soil sample taken from Kimberley Thorn Bushveld. Secondly, the aim was to test the isolates obtained from the Kimberley Thorn Bushveld soil sample in order to further explore the ability of mucoralean fungi to utilise the above mentioned series of nitrogen sources and to grow at an a, of 0.955. In addition, selected mucoralean taxa occurring frequently in soil habitats were tested for the ability to survive elevated temperatures in soil. It was found that the following species of the Mucorales may be encountered in the arid soil of the Karoo; Actinomucor elegans, CunninghamelIa echinulata, MortierelIa isabellina, Mucor circinelloides, Rhizomucor species, Rhizopus oryzae Went. Prins. Geerl. and Rhizopus stolonifer. Future surveys would reveal if genera like Absidia, GongronelIa and Zygorrhynchus, which have been isolated from arid regions, also occur in Karoo soil. Representatives of mucoralean taxa occurring in arid Karoo soil were able to utilise organic as well as inorganic oxidised nitrogen sources. However, at the concentration tested in this study, nitrite was found to be toxic to representatives of CunninghamelIa, Mortierell8, Rhizomucor and Rhizopus. Nitrate could not be utilised by CunninghamelIa, MortierelIa, Rhizomucor and Rhizopus stolonifer. Whether this inability to utilise inorganic nitrogen sources would prevail 'during oligotrophic growth in soil, remains a question to be addressed by future research. Representatives of the above mucoralean taxa occurring in arid soil were able to survive 55°C for 14 h in soil.
Open Access
An isolation procedure for arachidonic acid producing mucoralean fungi
(University of the Free State, 1999-11) Paul, Ida; Botha, A.; Kock, J. L. F.
English: Soil plates with malt extract agar and an incubation temperature of 5°C were used to selectively isolate representatives of the genus MorfierelIa from Alti Mountain Grassland soil. Fungi in the soil sample able to grow under these conditions amounted to a total of 2640 colony forming units per gram soil. Circa 94% of the total fungal isolates represented MorfierelIa subgenus MorfierelIa. The rest of the colony-forming units consisted of Mucor isolates (6.0%) and higher fungi (1.5%). Subsequently, the total lipids were extracted from the MorfierelIa isolates after cultivation on malt extract gelatine (MEG). The lipids were methylated using trimethyl sulphonium hydroxide and the methylated fatty acids in the lipids were identified using gas chromatography. The percentage arachidonic acid [20:4(ω6)], relative to other cellular long-chain fatty acids, was calculated. All the MorfierelIa strains isolated at 5°C from the Alti Mountain Grassland soil sample were found to produce 20:4(ω6). In the next part of the study, the radial growth rate on MEG was determined at 5°C and 20°C, for these MorfierelIa strains originally isolated at 5°C from Alti Mountain Grassland soil. To compare the growth of these strains with growth of other MorfierelIa strains, the radial growth rate at 5°C and 20°C was also determined for culture collection strains of MorfierelIa isolated at 25°C from Dry Sandy Highveld Grassland soil. In addition, 20:4(ω6) production in the 25°C isolates of MorfierelIa on MEG at 20°C was compared with the production of 20:4(ω6) in the MorfierelIa strains originally isolated at 5°C from Alti Mountain Grassland soil. The results indicated that all of the strains of MorfierelIa subgehus MorfierelIa originally isolated at 25°C and 5°C,were psychrotrophic and hence capable of growth at 25 °C, 20°C and 5°C. The results further indicated that the low temperature isolation procedure would be as suitable to isolate 20:4(ω6) producing MorfierelIa from soil, than isolation procedures utilizing complex media and 25°C as incubation temperature. However, this initial screening for 20:4(ω6) production was conducted on cultures grown on solid media (MEG). Consequently, four strains of M. alpine, which produced the highest percentage 20:4(ω6), were selected for further testing. Two of these strains were originally isolated at 5 °C from Alti Mountain Grassland soil and two were culture collection strains originally isolated at 25 °C from Dry Sandy Highveld Grassland soil. A reference strain of M. alpina obtained from an internationally recognized culture collection was also included in these experiments. Arachidonic acid accumulation in the neutral lipids of submerged cultures, were determined using two liquid media commonly used for this purpose. The media were Glucose Yeast Extract (GY) medium, and Hansson and Dostalek (HD) medium. Lipid analyses were conducted by harvesting fungal biomass in the stationary phase using filtration. The biomass was freeze dried and the total lipids extracted. The lipids were fractionated using column chromatography and the fatty acids of the neutral lipids were analyzed as methyl esters using gas chromatography. The volumetric concentration of 20:4(ω6) in the neutral lipids of each culture was subsequently calculated. The accumulation of 20:4(ω6) in M. alpina, originally isolated from Alti Mountain Grassland soil at 5 °C, was comparable to 20:4(ω6) accumulation in the reference strain obtained from an internationally recognized culture collection. However, the highest volumetric concentration of 20:4(ω6) in the neutral lipids obtained with the 5 °C isolates, was significantly lower than the highest volumetric concentration of 20:4(ω6) in the neutral lipids of M. alpina strains isolated at 25 °C from Dry Sandy Highveld Grassland soil. These results, therefore indicate that the low temperature isolation procedure, utilizing malt extract agar and an incubation temperature of 5 °C is not suitable for the isolation of MortierelIa strains producing high volumetric concentrations of 20:4(ω6) in the neutral lipids. However, it should be borne in mind that only five MortierelIa strains were tested for 20:4(ω6) production in this second part of the study. To confirm these results many more strains isolated at 5 °C and 25 °C from different soil habitats should be tested for 20:4(ω6) production in submerged cultures.
Open Access
Changes in microbial ecology during poultry production
(University of the Free State, 2000-03) Schreuder, Johanna Catharina; Viljoen, B. C.; Von Holy, A.; Cox, J.
Microorganisms, especially pathogens, play an important role in the deterioration of poultry meat and its products causing spoilage or food poisoning. The meat renders an ideal medium for the growth and progression of microorganisms originating from the environment on the farm (eggs and broiler), transport to the abattoir as well as the abattoir. As far as we know, no attempts were undertaken to determine the total microbial spectrum, including bacterial and yeast populations from the egg unto the chicken carcass after slaughtering. A historical review of the incidence and extent of microorganisms associated with poultry and it's environment is given in Chapter 1. The background of poultry production and contribution of microorganisms (for example pathogens and yeasts) are highlighted. In Chapter 2 a survey was undertaken to determine the incidence, extent and serotypes of different pathogens on and in freshly laid eggs as well as incubated eggs in the breeder broiler hatchery. In addition, the number of viable cells of bacteria and yeasts were also determined. Microbial counts on the dirty eggs predominated. A major decline in microbial numbers on the egg shells, however, were observed after 18 days in the incubator. The deduction in bacterial populations present on the egg shells after 18 days in the incubator, is ascribed to the constant fogging of the environment inside the incubator with clinafarm. The irregular gathering of eggs as well as the dirty hands of the egg collectors could have been the major contributors of the high counts on these eggs. The only pathogens obtained were Listeria and E. coli type 1.In Chapter 3 the microbial populations associated with the caecum and liver of broilers as well as with the environment were determined. Anaerobic plate counts reflecting the dominance of bacterial populations were constantly the highest ascribed to the contents of the gut. Salmonella, Staphylococcus aureus and E. coli type 1 were isolated from the caecum, however no Listeria were isolated. Salmonella and E. coli type 1 were isolated from the liver. The high incidence of pathogens associated with the broilers is an indication of the pathogens that enter the abattoir. These levels of pathogens, further increased during transport to the abattoir.In Chapter 4 the incidence and extent of microbial populations associated with broiler meat and the environment in the abattoir were evaluated. All the pathogens present on the farm were also observed in the abattoir. The main reasons for the similarity in the incidence of pathogens on the farm and the abattoir were ascribed to the transport from the farm to the abattoir, the process of slaughtering, equipment surfaces in the abattoir and people handling the meat. Aerobic plate counts predominated on the neckskin samples, equipment surfaces as well as in the water and air of the abattoir. E. coli type 1 clearly predominated on the neckskin samples as well as on the equipment surfaces, followed by Staphylococcus (U/reus and that by Salmonella. No Listeria isolates was, however, isolated from either the broiler farm or the abattoir, but was indeed observed on the egg shells from the breeder farm. Although a pattern reflecting the incidence of pathogens could be establish between the broiler farm and the abattoir, no comparison could be made with the eggs from the breeder farm and hatchery. This may be blamed on the sampling of insufficient number of eggs, or the lack of multiple repetitions. Despite the inability to detect the primary sources of pathogens the distinct possibility remained that the broiler farm was the main contributor towards microbial contamination and infection. Therefore, more effort is needed to control the diseases and infections on broiler farms, because in an abattoir you only get out what you put in.
Open Access
A comparative study of proteolysis in cheddar cheese and yeast-inoculated cheddar cheese during ripening
(University of the Free State, 2000-05) Botma, Maryna; Osthoff, G.; Viljoen, B.C.
English: Proteolysis is regarded as the most important event in the ripening of Cheddar cheese to contribute to the development of flavour. The characteristic proteolysis of each type of cheese is brought about by the enzymes used in the manufacturing process, e.g. rennet, as well as enzymes from the specific microbial cultures used in each cheese type. The effect of an inoculated yeast, Oebaryomyces hansenii, and its enzymes on proteolysis in Cheddar cheese was investigated. Proteolysis and development of peptides of the yeast-inoculated Cheddar cheese was followed throughout the ripening process and compared to the proteolysis in a standard Cheddar cheese. In a sensorical comparative study, no difference was found by a consumer panel between the two types of cheese, nor was any of the two significantly preferred. An expert panel however, judged the yeast-inoculated Cheddar cheese to be bitter. The proteins and peptides from the cheeses were extracted and fractionated by virtue of differences in solubility and molecular size. Yeast-inoculated Cheddar cheese contained less water-soluble nitrogen indicating a difference in proteolysis between the two types of cheese. Urea-polyacrylamide gel electrophoresis of the waterinsoluble fraction indicated that in the yeast-inoculated cheese rennet hydrolysis of o.si-casein was increased with faster formation of one primary peptide, o.s1-1as well as (o.s1-CN(f102-.)), with little further hydrolysis. The ~-casein was hydrolyzed slowly but with several additional peptides occurring. Reversed-phase high performance liquid chromatography of the watersoluble fraction indicated that a different peptide profile was formed, with at least five unique peptides at high amounts.
Open Access
The utilisation of used and other fats by fungi
(University of the Free State, 2000-09) Bareetseng, Sechaba; Christov, L.; Kock, J. L. F.
English: In 1997, Jeffery and co-workers discovered that when Mucor circinelloides t. circinelloides CBS 108.16 was cultivated on 30g/1 sunflower oil and 10g/1 sodium acetate, an improved utilisation of the oil, doubling of the biomass production and enhancement of the intracellular polyunsaturated y-linolenic acid (GLA) content occurred as compared to when this fungus was cultivated on only 40g/1 sunflower oil as sole carbon source. Consequently, the aim of this study became to further explore this phenomenon (hypothesis) in selected members of the zygomycotan fungi as well as yeasts when cultivated on various fat and oil substrates in the presence and absence of acetate. The ultimate aim was to identify those taxa that can be further explored for the transformation of edible and tall oils to high value lipids in the presence and absence of acetate. In this study, similar trends were observed in Mucor circinelloides f circinelloides CBS 108.16 as that found by Jeffery et al when cultivated on sunflower oil in the presence and absence of acetate. A similar pattern was also found when this fungus was grown on used cooking oil and tall oil. The enhancing effect of acetate was not generally observed in the other fungi and on some other fats and oils tested. Most fungi, including the yeasts, could grow on fats and oils provided in the presence or absence of acetate. Exceptions to the rule were MorfierelIa alpina MUFS Mo058 and Lipomyces starkeyi CBS 1807 T that were unable to grow. Schizosaccharomyces pombe var. pombe CBS 0356 T could also not grow on linseed oil. The presence of acetate had in many cases a stimulatory effect on growth. In some cases, the addition of acetate had an inhibitory effect on growth. With a few exceptions, most mucoralean fungi, when cultivated on various fats and oils, became oleaginous (i.e. contain ≥ 20% lipids) in the presence or absence of acetate. However, when these fungi were cultivated on linseed ail in the presence or absence of acetate they were unable to accumulate more than 20% lipids according to biomass. Less yeasts became oleaginous when cultivated on various fat and oil substrates when compared to the mucoralean fungi tested. The non-oleaginous yeasts i.e. Kluyveromyces, Saccharomyces, Schizosaccharomyces, Schwanniomyces and Yarrowia became oleaginous when cultivated on various fats and oils in the presence of acetate. In most mucoralean fungi and yeasts, the presence of acetate had an effect on cellular lipid content. The enhancing effect of acetate addition on cellular lipid content was experienced especially on GongronelIa, Mucor circinelloides t. circinelloides CBS 108.16 and Thamnostylum when cultivated on various fats and oils. On the other hand, the addition of acetate had a negative effect on cellular lipid accumulation in some mucoralean fungi and yeasts. In general, both the mucoralean fungi and yeasts utilised different fats and oils [(i.e. containing saturated fatty acids (FAs) and polyunsaturated fatty acids (PUFAs)] as carbon sources in the presence or absence of acetate. MortierelIa and Lipomyces could not utilise any of these fats and oils. The presence of acetate had a positive effect on fat and oil utilisation by most mucaralean fungi and some yeasts. In most cases, a pH increase was observed probably due to acetic acid utilisation during cultivation. In the presence or absence of acetate, most mucoralean fungi and yeasts showed a high preference towards the utilisation of PUFAs present in the residual oil fractions. The addition of acetate had both a positive and negative effect on the degree of preference towards PUFAs in the residual lipids. In most cases (in both mucoralean fungi and yeasts) lower amounts of PUFAs in the cellular lipid fractions in the presence or absence of acetate were found when compared to the oil substrate fed. This is probably due to the utilisation of these FAs through β-oxidation for the production of energy. No general pattern was observed regarding the effect of acetate addition on cellular PUFA content. Many mucoralean fungi when cultivated on various fats and oils (except soap skimmings) could produce GLA in the presence or absence of acetate. All the yeasts in this study could not produce GLA when cultivated on any lipid substrate in the presence or absence of acetate. This is probably due to the lack of a ∆6 desaturase enzyme. The addition of acetate improved GLA production in most mucoralean fungi when cultivated on sunflower oil and used cooking oil. Those fungi capable of producing GLA in this study should now be explored in the transformation of edible fats and oils to high value lipids containing GLA.
Open Access
An epidemiological survey of Newcastle disease virus in South Africa
(University of the Free State, 2001-05) Mashope, Barbara Keitumetse; Albertyn, J.; Bragg, R. R.; Van Heerden, E.
English:The primary objective in this study was the investigation of the molecular epidemiology of NDV strains isolated during epizooties in South Africa in 1998. Isolates purported to be NDV were collected from the Onderstepoort Veterinary Institute, the University of Pretoria, and EarlyBird Farms, Standerton. Four of the twenty-two isolates collected were identified as NDV, and successfully grouped into genotype VIIb, previously described by Herezeg et al., (1999). The isolates were propagated in 9-10 day-old embryonated SPF eggs. Embryo mortality was observed, allantoic fluid harvested, and genomic RNA extracted using TRIZOL ® reagent, containing guanidine thiocyanate. An RT-PCR based detection method was used to screen the isolates received (Ballagi- Pordány et al., 1996). The optimised method entailed initial reverse transcription in a reaction mixture containing IIlM random hexamers p(dN)6, 200 U Moloney Murine Leukemia (M-ML V) RT and 25 U RNasin. Aliquots of 5 ul, of the cDNA mix were used as template in subsequent PCR amplification reactions. A 1349bp region of the fusion protein gene was amplified using primers ONDVlaa and ONDV 4aa. Fusion protein amplicons were obtained from field isolates, M89/98 (Ost Pool I), M89/98 (Av Pool 2), M57/98, and M308/98 obtained from OP. Restriction enzyme profiles of the fusion protein amplicons using restriction enzymes HinfI, BstO I, and Rsa I displayed fragment patterns that allowed their grouping into genotype VIIb, described by Herezeg et al., (1999). Non-group specific bands observed upon digestion of the amplicon of strain M308/98 with Hinf I released fragments not consistent with those observed in genotype VIIb isolates. These fragments suggest the presence of mutations in this area of the genome. Amplification of a region comprising 88% of the matrix protein gene was facilitated using primers Ml and M2. These amplicons were subjected to RE analysis using restriction enzymes Mbt) I and Hinf I. Analysis of the restriction profiles produced revealed that these four strains were not re-isolated forms of the commonly used live vaccine LaSota strain. A region of the fusion protein gene encoding the fusion protein cleavage activation site was amplified by means of a nested peR, and sequenced. Initial peR amplified a region spanning from the M gene nt 778 to F gene nt 545 using primers Kl and K2. These amplicons were purified and used as template in a nested peR using primers MV1 (M gene nt 1163) and B2 (F gene nt 470). Sequence analysis revealed that the amino acid sequence at the fusion protein cleavage site of strains M89/98 (Ost Pool I), M57/98,and M308/98 was RRQKR -l-F, indicative of velogenie strains. Strain M89/98 (Av Pool 2) displayed a sequence at the fusion protein cleavage site that is characteristic of lentogenie strains GRQGR-l-L. This finding suggests that genotype VIIb isolates are heterogeneous, composed of strains of varying pathogenicity . Phylogenetic analysis based on a 378nt long region of the nested peR amplicon allowed the grouping of all these isolates into genotype VIIb. A 1.5-1% divergence was observed between group VIIb isolates collected in 1993, and 1995, and those used in this study, collected in 1998. This genetic distance is consistent with a 0.5-1% divergence in strains per year. The remaining field strains not detected by RT-peR, but that displayed embryo mortality indicative of pathogenic agents were subjected to HAlHI tests. Strains M193/98 (Ost Pool 6), M183/98 (Ost Liv B), and M193/98 (Ost Pool 5) agglutinated chicken erythrocytes. Haemagglutination was not inhibited by anti-NDV serum. These results led to the suspicion of the presence of an unidentified pathogen, most likely avian influenza, as all three samples were collected from ostrich hosts, from which this virus has previously been isolated in South Africa.
Open Access
Determination of the malting and milling performance of sorghum cultivars
(University of the Free State, 2001-05) Van Loggerenberg, Magdalena; Osthoff, G.; Pretorius, A. J.
English: Consumer demands for sorghum food acceptability stress the need for quality evaluation of milled and malted products. The aim of this study was to determine the milling and malting quality of sorghum cultivars with different physical and chemical qualities. The malting, physical and milling qualities of 24 sorghum cultivars were investigated over 2 seasons at 3 localities per season. The most important malting quality parameters that were identified with the canonical variate analysis were free amino nitrogen, diastatic power, germinative energy and vigour, compared to polyphenols, malting loss and water absorption that was identified as less important. From the canonical variate analysis, the most important physical qualities measured were sieve . fraction large, hundred kernel weight, water absorption and moisture content and were used to predict milling quality, while sieve fraction medium and small and meal fractions were found to be less important. Food quality properties were determined from roller and abrasive decortication milled meal. The abrasive hardness index, dehulling index and viscosity were important abrasive decortication milling parameters, with break 2 bran, break 1 bran, L-values, extraction, break 2 meal, break 1 meal and break 2 grits being important roller milling parameters. The best malting cultivars were APN 881, NK 286, PAN 8446, PAN 8660, SNK 3443, SNK 3337, SNK 3663, SNK 3939, SNK 3975, PAN 8564, NS 5655, SNK 3860 and NS 5511. Cultivars with the best physical properties were SNK 3939, SNK 3663, PAN 8061, PAN 8564, PAN 8171, SNK 3443, PAN 8446, SNK 3337, NK 286, APN 881, SNK 3883, SNK 3975, SNK 3567, PAN 8272 and PAN 8370. Good abrasive decortication milling property cultivars were APN 881, SNK 3567, SNK 3863, NK 286, ADV 5010, PAN 8564, SNK 3443, PAN 8370 and PAN 8660. Good roller milling cultivars were SNK 3883, SNK 3939, PAN 8564, NK 286 and SNK 3975. Cultivars with multi-functions were NK 286, PAN 8564 APN 881, SNK 3443, PAN 8660, SNK 3939, NS 5655 and SNK 3975 (good malting and milling).
Open Access
Participatory development of an indigenous goat cheese product: monitoring of the chemical, nutritional and microbiological quality from milk to cheese
(University of the Free State, 2001-05) Habteyohannes, Efrem Ghebremeskel; Hugo, C. J.; Roets, M.
English: The milk of indigenous goats has great potential in providing a nutritional food for poverty stricken communities. The aim of this study was to investigate the microbiological (spoilage and pathogenic), chemical (fat, protein and lactose) and nutritional (minerals, vitamins A and E) quality of indigenous goat milk from Mpumalanga and North West and compare it to Saanen goat milk. In almost all the quality parameters, the milk of the indigenous goats had a higher quality than the Saanen milk. The differences of most of the quality parameters between the indigenous and Saanen goat milk were ascribed to feed, breed, lactation stage, age, health status and the fact that the Saanen goats were machine-milked, while the indigenous goats were milked by hand. Another aim was to produce a Gouda-type cheese from each of the three goat milks. The microbiological (spoilage and pathogenic), chemical (moisture, total solids, ash, water activity, salt, fat content, free fat in dry matter, total free fatty acids, rancidity [TBA-value], fatty acid composition, protein content and proteolysis), nutritional (minerals and vitamins A and E) and sensory quality of the cheeses were then determined. The milk of the North West indigenous goats was acidic at the start of cheese production, but was normalized during production. The acidic nature of the cheese had, however, a negative effect on most of the quality parameters and resulted in a harder cheese with a goaty smell. The Mpumalanga and Saanen cheeses compared well in all the quality parameters and the sensory analysis suggested that no significant difference were present between these two cheeses. The conclusion was drawn that the milk of indigenous goats are an excellent way to provide a nutritional quality food, expand commercial markets, create jobs and increase income in rural areas.
Open Access
Ophiostoma species from hardwood sources in South Africa
(University of the Free State, 2001-10) De Beer, Zacharias Wilhelmus; Wingfield, M. J.; Wingfield, B. D.
English: The ophiostomatoid fungi are an economically important group of fungi, known for their ability to stain sapwood and cause tree diseases. In recent years, certain species in the group have also been considered as potential biological control agents in the pulp and paper industry. White mutants of species like Ophiostoma piliferum utilize pitch, which can cause problems in the pulping process, in freshly cut pulpwood chips. At the same time, other degrading fungi are out-competed. The first chapter of the thesis reviews the development, application, benefits and possible problems, of these biological control products. The possible application of such products in the South African pulp industry is also considered. One of the major concerns for the application of a biological control product such O. piliferum in South Africa, is the fact that it consists of a living fungus originating in the USA. A survey was, therefore, conducted to determine whether the fungus occurs in South Africa. The typical niche for O. piliferum is stained logs, lumber, and pulpwood chips. Isolates resembling O. piliferum were obtained from both exotic and indigenous wood sources. Based on morphology, these isolates could be separated into three groups, which resembled the descriptions of O. stenoceras, O. pluriannulatum, and O. piceae, respectively. For correct identification, the South African isolates had to be compared with herbarium material and authentic isolates from other parts of the world. The comparative taxonomic studies for the three groups of fungi form the basis of Chapters 4, 5 and 6 of this thesis. The taxonomic history of the genera Ophiostoma and Ceratocystis is complicated and confused. Published literature on the two genera, with the emphasis on Ophiostoma and its associated anamorph genera, is reviewed in Chapter 3. This serves as a background for the four following chapters of the thesis. Ribosomal DNA sequencing confirmed that one group of South African isolates is the same as O. stenoceras isolates from the Northern Hemisphere. Similar isolates from Colombia, Uruguay, and Kenya, were also included in the study and represent the first reports of O. stenoceras from these countries. Ophiostoma albidum, O. abietinum and O. nigrocarpum, all closely resemble O. stenoceras morphologically. Our sequence data show that O. albidum should be considered a synonym of O. stenoceras, and that O. abietinum is a synonym of O. nigrocarpum, which is a species distinct from O. stenoceras. For the past three decades, O. stenoceras has been considered the teleomorph of Sporothrix schenckii, the human pathogen. Our results, however, showed that rDNA sequences of the two species are significantly different, confirming that S. schenckii is a distinct species. The group of South African isolates resembling O. pluriannulatum, differed from this Northern Hemisphere species in that isolates have light brown perithecial bases and clubshaped ornamental hyphae on the perithecial bases. Similar isolates were obtained from Equador and Indonesia. Ribosomal DNA sequence data made it possible to distinguish between the two groups, and the Southern Hemisphere fungus is, therefore, described as a new species, Ophiostoma tropieale. Ophiostoma piceae and O. querei are virtually indistinguishable based on morphology, but hosts, rDNA sequences, and mating compatibility, can be used to separate the two species. By applying these criteria, the South African isolates resembling O. piceae obtained in the survey (Chapter 2), grouped with O. querei. Also included in the O. querei group were isolates from Brazil and Japan. One South African isolate, however, were identified as O. floccosum, representing the first report of this fungus from South Africa. The presence and distribution of species of the O. piceae complex in the Southern Hemisphere, are also discussed in Chapter 6. In recent literature, some confusion has emerged regarding the use of the name O. querei as opposed to 0. quercus. The last chapter of the thesis presents a brief review of the Latin and nomenclatural guidelines applicable in this particular case. The conclusion is that both names are grammatically acceptable. However, following the Code of Botanical Nomenclature, O. querei should be given preference. The work presented in this thesis contributes significantly to our understanding of the ophiostomatoid fungi, and in the greater context, biodiversity, in South Africa. Ribosomal DNA sequencing was successfully applied in conjunction with traditional taxonomic criteria to distinguish between species. However, many new questions arose from these results, especially regarding the phylogeny of Ophiostoma spp. with Sporothrix anamorphs. The results obtained from this study, will serve as the foundation for future research addressing these questions.
Open Access
Application of fungi in biotechnological processes for the pulp and paper industry
(University of the Free State, 2001-11) Dunn, Carin; Wolfaardt, J. F.
English: Lignin, hemicellulose and cellulose occur together in wood and agricultural wastes that are used in industries such as the pulp and paper industry. Biodegradation could be applied by these industries to save cost and reduce environmental impact. Lignocellulose degradation is very complex and must be understood for the optimisation of biotechnological processes. White-rot fungi degrade lignin, cellulose and hemicellulose while brown-rot fungi modify lignin slightly, but also break down cellulose and hemicellulose. The most important enzymes in lignin degradation are manganese peroxidase, lignin peroxidase and laccase, which these fungi produce extracellularly. Decay fungi establish on organic material, which leads to the degradation of lignocellulose during the colonization process. A distinction must be made between primary and secondary colonisers during degradation, because of succession that takes place. Ophiostoma piliferum is an example of a primary coloniser, which is used to produce Cartapip 9if). This product was developed to treat wood chips during storage to reduce fibre degradation. The fungus was previously not available in South Africa because of a possible threat to local forest species. South African forestry companies wanted to test Cartapip 97® in industrial processes and it was, therefore, necessary to demonstrate that the fungus is not pathogenic. It also had to be confirmed that the fungus is a strain of 0. piliferum before certification and importation into South Africa would be allowed. Cultural and morphological characteristics of the anamorph of the Cartapip fungus were found to be similar to those of O. piliferum and it was released for field trials. The pathogenicity of the fungus was compared with Ophiostoma ips and Sphaeropsis sapinea, causes of sapstain on Pinus spp. in South Afiica, to demonstrate that O. piliferum does not pose a threat to forestry. Different pine species were inoculated and the results indicated that O. piliferum is not a pathogen and it is, therefore, safe to use Cartapip 9if) in South Afiica. Hardwood and softwood chips were subsequently treated with Cartapip 97® and pulped using different pulping methods, but the benefits were not obvious. It is, however, possible that the extractives content of the chips was reduced. A slight increase in strength of kraft pulp from softwood and hardwood and also of Soda-AQ pulp from A. mearnsii, was observed after pre-treatment with Cartapip 97®. Bagasse contains fibres that can be used for the production of paper. However, bagasse has to be stored for long periods during which time decay occurs. Stored bagasse could be pre-treated with fungi to preserve and possibly improve the quality of the bagasse. In this study, Lenzites betulina and Pycnoporus sanguineus were used to treat bagasse before pulping. Inoculum production, pulping processes and different incubation periods were evaluated to optimise biopulping. Ultrastructural studies of treated bagasse were used to determine the effect that fungal treatment has on bagasse. Pulping results obtained from bagasse treated with L. betulina were variable and P. sanguineus did not improve the pulping. These results indicated that colonisation and degradation strategies of biopulping fungi must be fully understood before an attempt is made to optimize pulping processes.
Open Access
Cloning of the XynA gene from Thermomyces lanuginosus and expression in Saccharomyces cerevisiae
(University of the Free State, 2001-11) Nel, Sanet; Albertyn, J.; Van Heerden, E.; Bragg, R. R.
English: The xylanase from Thermomyces lanuginosus (XynA) was cloned into two shuttle vectors, pRS416 (single copy vector) and pRS426 (multi-copy vector) adjacent to a PDC1 promoter (designated pRS416:XynA and pRS426:XynA). An expression cassette for this xylanase was constructed by cloning of the XynA gene into a modified a-agglutinin (Aga1) gene from Saccharomyces cerevisiae. This modification entailed the deletion of the binding domain coding region of the Aga1, and the cloning of the XynA gene into this deleted binding domain region, which is flanked by a stalk-like protein coding region. This fusion protein was cloned into two shuttle vectors (pRS416 and pRS426), flanking the PDC1 promoter (designated pRS416:Aga1::XynA and pRS426:Aga1::XynA). The aim of the cassette was to immobilize the expressed enzyme on the cell surface of the yeast cell with the expression of the xylanase on the stalk of the Aga1, however, extracellular secretion of the enzyme was obtained upon expression. Enzyme assays performed on pRS416:XynA and pRS426:XynA yielded very low activity [0.1505 U/ml (2.5088 nKat/ml) and 0.0909 U/ml (1.5153 nKat/ml) respectively], whereas pRS416:Aga1::XynA and pRS426:Aga1::XynA yielded activities of 1.7035 U/ml (28.3973 nKat/ml) and 1.7319 U/ml (28.8707 nKat/ml) respectively. The partial characterization of this extracellular secreted recombinant xylanase (pRS416:Aga1::XynA and pRS426:Aga1::XynA) yielded an optimum temperature of 70 °C and an optimum pH of 6.0-7.0. Thermal stability for the recombinant xylanase was determined for temperatures 50 °C, 60 °C and 70 °C, and the activation energy for pRS416:Aga1::XynA and pRS426:Aga1::XynA were calculated as 34.86 kJ/mol and 53.59 kJ/mol respectively.