The isolation of gamma-linolenic acid producing mucoralean fungi

Abstract

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.

Afrikaans: Lede van Mucorales is bekend vir hul vermoë om die hoë waarde vetsuur, gamma-linoleensuur 18:3(ω6) te produseer. Hoewel daar slegs enkele studies gedoen is, is dit bekend dat die tipe koolstofbron in die medium die produksie van 18:3(ω6) deur die fungi beïnvloed. Die spektrum koolstofbronne waarop mucoraliese fungi kan groei en 18:3(ω6)) produseer, IS egter nog grootliks onbekend. Nog'n faktor wat die hoeveelheid 18:3(ω6) wat deur die fungi geproduseer word beïnvloed, is die spesifieke fungusstam wat in die proses gebruik word. Daar is gevolglik besluit om in hierdie studie eers ondersoek in te stel na die vermoë van die mucoraliese fungi om op 'n reeks koolstofbronne te groei en 18:3(ω6) te produseer. Daarna sal isolasiemedia ontwerp word waarmee nuwe fungusstamme wat koolstofbronne teenwoordig in industriële aflope kan benut, uit die natuur geïsoleer kan word. Die invloed van 38 verskillende koolstofbronne op groei en gevolglike 18:3(ω6)) inhoud van die lipiede geproduseer deur vier mucoraliese funqusstamme, is ondersoek. Die stamme het die spesies MortierelIa alpina, Mucor circinelloides, Mucor flavus en Thamnosfylum piriforme verteenwoordig. Die verteenwoordigers van M. circinelloides en M. flavus kon onderskeidelik 25 en 23 van die 38 koolstofbronne benut. Die hoogste persentasies 18:3(ω6) wat verkry is met die verteenwoordigers van M. circinelloides en M. flavus, was 27.17 % en 36.40 % onderskeidelik. In teenstelling hiermee, was die hoogste persentasies 18:3(ω6) wat verkry is met die verteenwoordigers van Mo. alpina en T. piriforme slegs 5.61 % en 12.84 % onderskeidelik. Hierdie twee stamme kon slegs onderskeidelik sewe en 17 van die koolstofbronne benut. Hierdie studie is dus 'n aanduiding dat mucoraliese fungi kan groei en 18:3(ω6) produseer op 'n wye verskeidenheid van koolstofbronne, insluitende koolstofbronne teenwoordig in industriële aflope (bv. stysel, sukrose en asynsuur). Drie selektiewe media is hierna ontwikkel om mucoraliese fungi vanuit grond te isoleer d.m.v. die grondplaattegniek. Die media, wat kompleks is, het onderskeidelik stysel, sukrose en natrium asetaat as koolstofbronne bevat, asook 0.02 g/I van die anti-fungus middel, benlate. Die selektiwiteit van die media vir Mucorales is bepaal deur die media se vermoë te toets om groei te ondersteun van 134 mucoraliese fungus stamme, wat 66 spesies en sewe genera verteenwoordig. Die drie isolasiemedia het groei van die stamme van Absidia, Actinomucor, Backusella, Mucor, Rhizopus en Thamnosty/um ondersteun. Die vermoë van die isolasiemedia om vir mucoraliese fungi in 'n natuurlike fungus populasie in grond te selekteer, is bepaal. Verteenwoordigers van die genera Absidia, CunninghamelIa, GongronelIa, Mucor en Rhizopus is verkry. Die resultate het verder bewys dat deur selektiewe media in kombinasie met 'n relatiewe nie-selektiewe medium te gebruik, i.p.v. die nie-selektiewe medium alleen, meer mucoraliese taksa geïsoleer kan word vanaf 'n spesifieke grondmonster. Mucoraliese fungusisolate wat uit die grondmonster verkry is, is gevolglik geëvalueer vir groei en 18:3(ω6)produksie in media wat stysel, sukrose of glukose as koolstofbronne bevat. Isolate wat die families Absidiaceae, Cunninghamellaceae en Mucoraceae verteenwoordig is geïnokuleer in komplekse media wat die bogenoemde koolstofbronne bevat. Daar is gevind dat al die isolate 18:3(ω6)kon produseer op al drie koolstofbronne, hoewel betekenisvolle verskille in die volumetriese 18:3(ω6)konsentrasies verkry is op die verskillende koolstofbronne vir elke isolaat wat getoets is. Die hoogste volumetriese konsentrasies van 18:3(ω6)is verkry van die isolaat wat R. stoloniter verteenwoordig. Stysel (0.130 g/I) en glukose (0.134 g/I) is as koolstofbronne gebruik. Om te bewys dat die isolate wat verkry is van die bogenoemde isolasiemedia in 'n industriële afloop kan groei, is sommige van die isolate wat verskillende families verteenwoordig, opgegroei in 'n medium wat voorberei is van 'n industriële afloop wat dekstriene, galaktane en stysel as koolstofbron bevat. Die lipiede van die isolate wat die CSB waarde van die afloop die meeste verminder het, is geanaliseer. Dit is gevind dat die isolate 18:3(ω6) kon produseer. Die studie het dus bewys dat dit moontlik is om isolasiemedia te ontwerp om 18:3(ω6) produserende mucoraliese fungi uit 'n natuurlike fungus populasie te isoleer. Dit is ook gevind dat die isolate gebruik kan word om biomassa en 18:3(ω6)te produseer van koolstofbronne wat in industriële aflope teenwoordig is.