An isolation procedure for arachidonic acid producing mucoralean fungi

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.