The isolation of gamma-linolenic acid producing mucoralean fungi

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.