An isolation procedure for arachidonic acid producing mucoralean fungi

[摘要] English: Soil plates with malt extract agar and an incubation temperature of 5°C were used toselectively isolate representatives of the genus MorfierelIa from Alti Mountain Grasslandsoil. Fungi in the soil sample able to grow under these conditions amounted to a totalof 2640 colony forming units per gram soil. Circa 94% of the total fungal isolatesrepresented MorfierelIa subgenus MorfierelIa. The rest of the colony-forming unitsconsisted of Mucor isolates (6.0%) and higher fungi (1.5%). Subsequently, the totallipids were extracted from the MorfierelIa isolates after cultivation on malt extractgelatine (MEG). The lipids were methylated using trimethyl sulphonium hydroxide andthe methylated fatty acids in the lipids were identified using gas chromatography. Thepercentage 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 MountainGrassland 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 MorfierelIastrains originally isolated at 5°C from Alti Mountain Grassland soil. To compare thegrowth of these strains with growth of other MorfierelIa strains, the radial growth rate at5°C and 20°C was also determined for culture collection strains of MorfierelIa isolatedat 25°C from Dry Sandy Highveld Grassland soil. In addition, 20:4(ω6) production inthe 25°C isolates of MorfierelIa on MEG at 20°C was compared with the production of20:4(ω6) in the MorfierelIa strains originally isolated at 5°C from Alti Mountain Grasslandsoil. The results indicated that all of the strains of MorfierelIa subgehus MorfierelIaoriginally isolated at 25°C and 5°C,were psychrotrophic and hence capable of growthat 25 °C, 20°C and 5°C. The results further indicated that the low temperature isolationprocedure would be as suitable to isolate 20:4(ω6) producing MorfierelIa from soil, thanisolation procedures utilizing complex media and 25°C as incubation temperature.However, this initial screening for 20:4(ω6) production was conducted on culturesgrown on solid media (MEG). Consequently, four strains of M. alpine, which producedthe highest percentage 20:4(ω6), were selected for further testing. Two of these strainswere originally isolated at 5 °C from Alti Mountain Grassland soil and two were culturecollection strains originally isolated at 25 °C from Dry Sandy Highveld Grassland soil. Areference strain of M. alpina obtained from an internationally recognized culturecollection was also included in these experiments. Arachidonic acid accumulation inthe neutral lipids of submerged cultures, were determined using two liquid mediacommonly used for this purpose. The media were Glucose Yeast Extract (GY) medium,and Hansson and Dostalek (HD) medium. Lipid analyses were conducted by harvestingfungal biomass in the stationary phase using filtration. The biomass was freeze driedand the total lipids extracted. The lipids were fractionated using columnchromatography and the fatty acids of the neutral lipids were analyzed as methyl estersusing gas chromatography. The volumetric concentration of 20:4(ω6) in the neutrallipids of each culture was subsequently calculated. The accumulation of 20:4(ω6) inM. alpina, originally isolated from Alti Mountain Grassland soil at 5 °C, was comparableto 20:4(ω6) accumulation in the reference strain obtained from an internationallyrecognized culture collection. However, the highest volumetric concentration of20:4(ω6) in the neutral lipids obtained with the 5 °C isolates, was significantly lowerthan the highest volumetric concentration of 20:4(ω6) in the neutral lipids of M. alpinastrains isolated at 25 °C from Dry Sandy Highveld Grassland soil. These results,therefore indicate that the low temperature isolation procedure, utilizing malt extractagar and an incubation temperature of 5 °C is not suitable for the isolation ofMortierelIa strains producing high volumetric concentrations of 20:4(ω6) in the neutrallipids. However, it should be borne in mind that only five MortierelIa strains were testedfor 20:4(ω6) production in this second part of the study. To confirm these results manymore strains isolated at 5 °C and 25 °C from different soil habitats should be tested for20:4(ω6) production in submerged cultures.

[发布机构] University of the Free State