Masters Degrees (Microbial, Biochemical and Food Biotechnology)

Permanent URI for this collection

http://hdl.handle.net/11660/447

Browse

Browsing Masters Degrees (Microbial, Biochemical and Food Biotechnology) by Author "Botha, A."

Now showing 1 - 3 of 3
  • Thumbnail Image
    ItemOpen 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.
  • Thumbnail Image
    ItemOpen 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.
  • Thumbnail Image
    ItemOpen 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.