An isolation procedure for arachidonic acid producing mucoralean fungi
| dc.contributor.advisor | Botha, A. | |
| dc.contributor.advisor | Kock, J. L. F. | |
| dc.contributor.author | Paul, Ida | |
| dc.date.accessioned | 2017-06-06T06:10:20Z | |
| dc.date.available | 2017-06-06T06:10:20Z | |
| dc.date.issued | 1999-11 | |
| dc.description.abstract | 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. | en_ZA |
| dc.description.abstract | Afrikaans: Grondplate met moutekstrak-agar en 'n inkubasietemperatuur van 5°C is gebruik om verteenwoordigers van die genus MorfierelIa selektief uit Alti Berg Grasland grond te isoleer. Fungi in die grondmonster wat in staat was om onder hierdie kondisies te groei het 'n totaal van 2640 kolonievormende eenhede per gram grond beloop. Circa 94% van die totale fungusisolate het MorfierelIa subgenus MorfierelIa verteenwoordig. Die res van die kolonievormende eenhede het uit Mucor isolate (6.0%) en hoër fungi (1.5%) bestaan. Vervolgens is die totale lipiede uit die MorfierelIa isolate geëkstraheer ná kweking op moutekstrak-gelatien (MEG). Die lipiede is gemetileer deur middel van trimetiel-sulfonium-hidroksied en die gemetileerde vetsure in die lipiede is deur middel van gaschromatografie geïdentifiseer. Die persentasie arachidonsuur [20:4(ω 6)] relatief tot ander sellulêre langketting-vetsure is vervolgens bereken. AI die MorfierelIa stamme wat by 5°C uit die Alti Berg Grasland grondmonster geïsoleer is, is gevind om 20:4(ω 6) te produseer. In die volgende gedeelte van die studie is die radiale groeitempo op MEG by 5°C en 20°C bepaal vir hierdie MorfierelIa stamme wat oorspronklik by 5°C uit Alti Berg Grasland grond geïsoleer is. Om die groei van hierdie stamme met die groei van ander MorfierelIa stamme te vergelyk, is die radiale groeitempo van MorfierelIa stamme wat by 25 °C uit Droë Sanderige Hoëveld Grasland grond geïsoleer is, ook by 5 °C en 20 °C bepaal. Verder is die 20:4(ω6)produksie in die 25 °C isolate van MorfierelIa op MEG by 20 °Cvergelyk met die produksie van 20:4(ω6)in die MortierelIa stamme wat oorspronklik by 5 °C uit die Alti Berg Grasland grond geïsoleer is. Die resultate het aangedui dat al die stamme van MortierelIa subgenus MorfierelIa wat oorsponklik by 25 °C en 5 °C geïsoleer is, psigrotolerant was en dus in staat tot groei by 25 °C, 20 °C en 5 °C. Die resultate het verder aangetoon dat die laer temperatuur isolasieprosedure net so geskik sal wees vir die isolasie van 20:4(ω6) produserende Morfiere/la uit grond as isolasieprosedures wat komplekse media en 25 °C as inkubasietemperatuur gebruik. Hierdie aanvanklike sifting vir 20:4(ω6) produksie is uitgevoer op kulture wat op soliede media gekweek is (MEG). Gevolglik is vier stamme van M. alpina, wat die hoogste persentasie 20:4(ω6) geproduseer het, vir verdere toetsing geselekteer. Twee van hierdie stamme is oorspronklik by 5°C uit Alti Berg Grasland grond geïsoleer, en twee was kultuurversamelingstamme, wat oorsronklik uit Droë Sanderige Hoëveld Grasland grond geïsoleer is. 'n Verwysingstam, afkomstig vanaf 'n internasionaal erkende kultuurversameling was ook in hierdie eksperimente ingesluit. Arachidonsuurakkumulasie in die neutrale lipiede van onderdompelde kulture is bepaal deur gebruik te maak van twee vloeibare media wat algemeen vir hierdie doel gebruik word. Die media was Glukose Gis ekstrak (GG) en Hansson en Dostalek medium (HD). Lipiedanalises is uitgevoer deur die fungusbiomassa tydens die stasionêrefase deur middel van filtrasie te oes. Die biomassa is vervolgens gevriesdroog en die totale lipiede is geëkstraheer. Die lipiede is gefraksioneer deur kolom-chromatografie en die vetsure van die neutrale lipiede is as metielesters geanaliseer deur middel van gaschromatografie. Die volumetriese konsentrasie van 20:4(ω6) in die neutrale lipiede van elke kultuur is gevolglik bereken. Daar is bevind dat 20:4(ω6) akkumulasie in M. alpina, oorspronklik geïsoleer by 5 °C uit Alti Berg Grasland grond, vergelykbaar was met 20:4(ω6) akkumulasie in die verwysingstam wat vanaf 'n internasionaal erkende kultuurversameling verkry was. Maar die hoogste volumetriese konsentrasie 20:4(ω6) in die neutrale lipiede wat met die 5 °C isolate verky is, was betekenisvol laer as die hoogste konsentrasie 20:4(ω6) in die neutrale lipiede van M. alpina stamme wat by 25 °C uit Droë Sanderige Hoëveld Grasland grond geïsoleer is. Die resultate dui aan dat die lae-ternperatuurs., isolasieprosedure, waar moutekstrak-agar en 'n inkubasietemperatuur van 5°C gebruik word, ongeskik is vir die isolering van Mortiere/la stamme wat hoë volumetriese konsentrasies 20:4(ω6) in die neutrale lipiede produseer. Dit moet egter in ag geneem word dat slegs vyf Mortiere/la stamme getoets was vir die produksie van 20:4(ω6) in die tweede gedeelte van hierdie studie. Om hierdie resultate te bevestig, moet meer stamme, wat by 5 °C en 25 °Cvanaf verskillende grondhabitatte geisoleer is, getoets word vir die produksie van 20:4(ω6) in onderdompelde kulture. | af |
| dc.description.sponsorship | National Research Foundation (NRF) | en_ZA |
| dc.identifier.uri | http://hdl.handle.net/11660/6334 | |
| dc.language.iso | en | en_ZA |
| dc.publisher | University of the Free State | en_ZA |
| dc.rights.holder | University of the Free State | en_ZA |
| dc.subject | Arachidonic acid | en_ZA |
| dc.subject | Fungi | en_ZA |
| dc.subject | Isolation | en_ZA |
| dc.subject | Lipids | en_ZA |
| dc.subject | MortierelIa | en_ZA |
| dc.subject | Soil | en_ZA |
| dc.subject | Fatty acids | en_ZA |
| dc.subject | Zygomycetes | en_ZA |
| dc.subject | Linolenic acids | en_ZA |
| dc.subject | Mucorales | en_ZA |
| dc.subject | Dissertation (M.Sc. (Microbiology and Biochemistry))--University of the Free State, 1999 | en_ZA |
| dc.title | An isolation procedure for arachidonic acid producing mucoralean fungi | en_ZA |
| dc.type | Dissertation | en_ZA |