Scholarly Work - Plant Sciences & Plant Pathology
Permanent URI for this collectionhttps://scholarworks.montana.edu/handle/1/8870
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Item Modulation of volatile organic compound formation in the Mycodiesel-producing endophyte Hypoxylon sp. CI-4(2012-11) Ul-Hassan, Syed Riyaz; Strobel, Gary A.; Booth, Eric; Knighton, W. Berk; Floerchinger, Cody; Sears, JoeAn endophytic Hypoxylon sp. (strain CI-4) producing a wide spectrum of volatile organic compounds (VOCs), including 1,8-cineole, 1-methyl-1,4-cyclohexadiene and cyclohexane, 1,2,4-tris(methylene), was selected as a candidate for the modulation of VOC production. This was done in order to learn if the production of these and other VOCs can be affected by using agents that may modulate the epigenetics of the fungus. Many of the VOCs made by this organism are of interest because of their high energy densities and thus the potential they might have as Mycodiesel fuels. Strain CI-4 was exposed to the epigenetic modulators suberoylanilide hydroxamic acid (SAHA, a histone deacetylase) and 5-azacytidine (AZA, a DNA methyltransferase inhibitor). After these treatments the organism displayed striking cultural changes, including variations in pigmentation, growth rates and odour, in addition to significant differences in the bioactivities of the VOCs. The resulting variants were designated CI4-B, CI4-AZA and CI4-SAHA. GC/MS analyses of the VOCs produced by the variants showed considerable variation, with the emergence of several compounds not previously observed in the wild-type, particularly an array of tentatively identified terpenes such as α-thujene, sabinene, γ-terpinene, α-terpinolene and β-selinene, in addition to several primary and secondary alkanes, alkenes, organic acids and derivatives of benzene. Proton transfer reaction mass spectroscopic analyses showed a marked increase in the ratio of ethanol (mass 47) to the total mass of all other ionizable VOCs, from ~0.6 in the untreated strain CI-4 to ~0.8 in CI-4 grown in the presence of AZA. Strain CI4-B was created by exposure of the fungus to 100 µM SAHA; upon removal of the epigenetic modulator from the culture medium, it did not revert to the wild-type phenotype. Results of this study have implications for understanding why there may be a wide range of VOCs found in various isolates of this fungus in nature.Item The Paleobiosphere: a novel device for the in vivo testing of hydrocarbon producing-utilizing microorganisms(2013-04) Strobel, Gary A.; Booth, Eric; Schaible, George A.; Mends, Morgan Tess; Sears, Joe; Geary, BradThe construction and testing of a unique instrument, the Paleobiosphere, which mimics some of the conditions of the ancient earth, is described. The instrument provides an experimental testing system for determining if certain microbes, when provided an adequate environment, can degrade biological materials to produce fuel-like hydrocarbons in a relatively short time frame that become trapped by the shale. The conditions selected for testing included a particulate Montana shale (serving as the “Trap Shale”), plant materials (leaves and stems of three extant species whose origins are in the late Cretaceous), a water-circulating system, sterile air, and a specially designed Carbotrap through which all air was passed as exhaust and volatile were hydrocarbons trapped. The fungus for initial testing was Annulohypoxylon sp., isolated as an endophyte of Citrus aurantifolia. It produces, in solid and liquid media, a series of hydrocarbon-like molecules. Some of these including 1,8-cineole, 2-butanone, propanoic acid, 2-methyl-, methyl ester, benzene (1-methylethyl)-, phenylethyl alcohol, benzophenone and azulene, 1,2,3,5,6,7,8,8a-octahydro-1,4-dimethyl-7-(1-methylethenyl), [1S-(1α,7α,8aβ)]. These were the key signature compounds used in an initial Paleobiosphere test. After 3 weeks, incubation, the volatiles associated with the harvested “Trap Shale” included each of the signature substances as well as other fungal-associated products: some indanes, benzene derivatives, some cyclohexanes, 3-octanone, naphthalenes and others. The fungus thus produced a series of “Trap Shale” products that were representative of each of the major classes of hydrocarbons in diesel fuel (Mycodiesel). Initial tests with the Paleobiosphere offer some evidence for a possible origin of hydrocarbons trapped in bentonite shale. Thus, with modifications, numerous other tests can also be designed for utilization in the Paleobiosphere.Item An endophytic Nodulisporium sp. producing volatile organic compounds having bioactivity and fuel potential(2012) Mends, Morgan Tess; Yu, Eizadora; Strobel, Gary A.; Hassan, S. R. U.; Booth, Eric; Geary, Brad; Sears, Joe; Taatjes, C. A.; Hadi, M.Nodulisporium sp. has been isolated as an endophyte of Myroxylon balsamum found in the upper Napo region of the Ecuadorian Amazon. This organism produces volatile organic compounds (VOCs) that have both fuel and biological potential.Under microaerophilic growth environments, the organism produces 1, 4-cyclohexadiene, 1 methyl-,1-4 pentadiene and cyclohexene, 1-methyl-4-(1-methylethenyl)- along with some alcohols and terpenoids of interest as potential fuels. The fungus was scaled up in an aerated large fermentation flask, and the VOCs trapped by Carbotrap technology and analyzed by headspace solid –phase microextraction (SPME) fiber-GC/MS. Under these conditions, Nodulisporium sp. produces a series of alkyl alcohols starting with 1-butanol-3-methyl, 1- propanol-2-methyl, 1- pentanol, 1-hexanol, 1-heptanol, 1- octanol, 1-nonanol along with phenylethyl alcohol.The organism also produces secondary alkyl alcohols, esters, ketones, benzene derivatives, a few terpenoids, and some hydrocarbons. It appears that many of the products have fuel potential. Furthermore, the VOCs of Nodulisporium sp. were active against a number of pathogens causing death to both Aspergillus fumigatus and Rhizoctonia solani and severe growth inhibition produced in Phytophthora cinnamomi and Sclerotinia sclerotiorum within 48 hr of exposure. The Carbotrapped materials somewhat mimicked the bioactivities of the culture itself when certain test organisms were exposed to these VOCs. A brief discussion on the relationship of these fungal VOCs to those compounds found in transportation fuels is presented.