Browsing by Author "Knighton, W. Berk"

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Endophytic fungal production rates of volatile organic compounds are highest under microaerophilic conditions
(2017-11) Schoen, Heidi R.; Knighton, W. Berk; Peyton, Brent M.
Volatile organic compound (VOC) production from an endophytic fungus was quantified at four oxygen concentrations (0, 1, 13 and 21 %) throughout culture growth phases. The filamentous fungus, a Nodulisporium sp. (designated TI-13), was grown in a solid-state reactor with an agricultural byproduct, beet pulp, as the solid substrate. The VOCs, with potential applications as biofuels, natural flavour compounds and bioactive mixtures, were measured with a recently introduced platinum catalyst and proton transfer reaction mass spectrometry quantification system. The highest-specific production rates of carbon number four and higher VOCs were observed under microaerophilic conditions, which is the expected environment within the plant host. Specific production rates of two ester compounds increased by at least 19 times under microaerophilic conditions compared with those under any other oxygen concentration studied. Total VOC production, including small molecules such as ethanol and acetaldehyde, increased by 23 times when compared between aerobic and anoxic conditions, predominately due to increased production of ethanol. Additionally, total specific production for all 21 compounds quantified was highest under reduced oxygen conditions.
Functionalized para-substituted benzenes as 1,8-cineole production modulators in an endophytic Nodulisporium species
(2014-08) Nigg, Jered; Strobel, Gary A.; Knighton, W. Berk; Hilmer, Jonathan K.; Geary, Brad; Ul-Hassan, Syed Riyaz; Harper, James K.; Valenti, Domenic J.; Wang, Yuemin
A Nodulisporium species (designated Ti-13) was isolated as an endophyte from Cassia fistula. The fungus produces a spectrum of volatile organic compounds (VOCs) that includes ethanol, acetaldehyde and 1,8-cineole as major components. Initial observations of the fungal isolate suggested that reversible attenuation of the organism via removal from the host and successive transfers in pure culture resulted in a 50 % decrease in cineole production unrelated to an overall alteration in fungal growth. A compound (CPM1) was obtained from Betula pendula (silver birch) that increases the production of 1,8-cineole by an attenuated Ti-13 strain to its original level, as measured by a novel bioassay method employing a 1,8-cineole-sensitive fungus (Sclerotinia sclerotiorum). The host plant produces similar compounds possessing this activity. Bioactivity assays with structurally similar compounds such as ferulic acid and gallic acid suggested that the CPM1 does not act as a simple precursor to the biosynthesis of 1,8-cineole. NMR spectroscopy and HPLC-ES-MS indicated that the CPM1 is a para-substituted benzene with alkyl and carboxyl substituents. The VOCs of Ti-13, especially 1,8-cineole, have potential applications in the industrial, fuel and medical fields.
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, Joe
An 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.
The production of myco-diesel hydrocarbons and their derivatives by the endophytic fungus Gliocladium roseum (NRRL 50072)
(2010-12) Strobel, Gary A.; Knighton, W. Berk; Kluck, Katreena; Ren, Yuhao; Livinghouse, Tom; Griffin, Meghan; Spakowicz, Daniel; Sears, Joe
An endophytic fungus, Gliocladiun roseum (NRRL 50072), produced a series of volatile hydrocarbons and hydrocarbon derivatives on an oatmeal-based agar under microaerophilic conditions as analysed by solid-phase micro-extraction (SPME)-GC/MS. As an example, this organism produced an extensive series of the acetic acid esters of straight-chained alkanes including those of pentyl, hexyl, heptyl, octyl, sec-octyl and decyl alcohols. Other hydrocarbons were also produced by this organism, including undecane, 2,6-dimethyl; decane, 3,3,5-trimethyl; cyclohexene, 4-methyl; decane, 3,3,6-trimethyl; and undecane, 4,4-dimethyl. Volatile hydrocarbons were also produced on a cellulose-based medium, including heptane, octane, benzene, and some branched hydrocarbons. An extract of the host plant, Eucryphia cordifolia (ulmo), supported the growth and hydrocarbon production of this fungus. Quantification of volatile organic compounds, as measured by proton transfer mass spectrometry (PTR-MS), indicated a level of organic substances in the order of 80 p.p.m.v. (parts per million by volume) in the air space above the oatmeal agar medium in an 18 day old culture. Scaling the PTR-MS profile the acetic acid heptyl ester was quantified (at 500 p.p.b.v.) and subsequently the amount of each compound in the GC/MS profile could be estimated; all yielded a total value of about 4.0 p.p.m.v. The hydrocarbon profile of G. roseum contains a number of compounds normally associated with diesel fuel and so the volatiles of this fungus have been dubbed ‘myco-diesel’. Extraction of liquid cultures of the fungus revealed the presence of numerous fatty acids and other lipids. All of these findings have implications in energy production and utilization.
Rapid total volatile organic carbon quantification from microbial fermentation using a platinum catalyst and proton transfer reaction-mass spectrometry
(2016-10) Schoen, Heidi R.; Peyton, Brent M.; Knighton, W. Berk
A novel analytical system was developed to rapidly and accurately quantify total volatile organic compound (VOC) production from microbial reactor systems using a platinum catalyst and a sensitive CO2 detector. This system allows nearly instantaneous determination of total VOC production by utilizing a platinum catalyst to completely and quantitatively oxidize headspace VOCs to CO2 in coordination with a CO2 detector. Measurement of respiratory CO2 by bypassing the catalyst allowed the total VOC content to be determined from the difference in the two signals. To the best of our knowledge, this is the first instance of a platinum catalyst and CO2 detector being used to quantify the total VOCs produced by a complex bioreactor system. Continuous recording of these CO2 data provided a record of respiration and total VOC production throughout the experiments. Proton transfer reaction-mass spectrometry (PTR-MS) was used to identify and quantify major VOCs. The sum of the individual compounds measured by PTR-MS can be compared to the total VOCs quantified by the platinum catalyst to identify potential differences in detection, identification and calibration. PTR-MS measurements accounted on average for 94 % of the total VOC carbon detected by the platinum catalyst and CO2 detector. In a model system, a VOC producing endophytic fungus Nodulisporium isolate TI-13 was grown in a solid state reactor utilizing the agricultural byproduct beet pulp as a substrate. Temporal changes in production of major volatile compounds (ethanol, methanol, cetaldehyde, terpenes, and terpenoids) were quantified by PTR-MS and compared to the total VOC measurements taken with the platinum catalyst and CO2 detector. This analytical system provided fast, consistent data for evaluating VOC production in the nonhomogeneous solid state reactor system.
Resolution of volatile fuel compound profiles from Ascocoryne sarcoides: A comparison by proton transfer reaction-mass spectrometry and solid phase microextraction gas chromatography mass spectrometry
(2012-04) Mallette, Natasha D.; Knighton, W. Berk; Strobel, Gary A.; Carlson, Ross P.; Peyton, Brent M.
Volatile hydrocarbon production by Ascocoryne sacroides was studied over its growth cycle. Gas-phase compounds were measured continuously with a proton transfer reaction-mass spectrometry (PTR-MS) and at distinct time points with gas chromatography-mass spectrometry (GC-MS) using head space solid phase microextraction (SPME). The PTR-MS ion signal permitted temporal resolution of the volatile production while the SPME results revealed distinct compound identities. The quantitative PTR-MS results showed the volatile production was dominated by ethanol and acetaldehyde, while the concentration of the remainder of volatiles consistently reached 2,000 ppbv. The measurement of alcohols from the fungal culture by the two techniques correlated well. Notable compounds of fuel interest included nonanal, 1-octen-3-ol, 1-butanol, 3-methyl- and benzaldehyde. Abiotic comparison of the two techniques demonstrated SPME fiber bias toward higher molecular weight compounds, making quantitative efforts with SPME impractical. Together, PTR-MS and SPME GC-MS were shown as valuable tools for characterizing volatile fuel compound production from microbiological sources.