WO2014165174A2 - Microorganismes pour la production de composés organiques volatils et procédés les utilisant - Google Patents

Microorganismes pour la production de composés organiques volatils et procédés les utilisant Download PDF

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WO2014165174A2
WO2014165174A2 PCT/US2014/024650 US2014024650W WO2014165174A2 WO 2014165174 A2 WO2014165174 A2 WO 2014165174A2 US 2014024650 W US2014024650 W US 2014024650W WO 2014165174 A2 WO2014165174 A2 WO 2014165174A2
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fungus
vocs
isolated
nodulisporium
compounds
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PCT/US2014/024650
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WO2014165174A3 (fr
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Gary A. Strobel
Angela R. Tomsheck
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Strobel Gary A
Tomsheck Angela R
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Priority claimed from US13/796,469 external-priority patent/US9090921B2/en
Priority claimed from US13/796,527 external-priority patent/US20130252313A1/en
Application filed by Strobel Gary A, Tomsheck Angela R filed Critical Strobel Gary A
Publication of WO2014165174A2 publication Critical patent/WO2014165174A2/fr
Publication of WO2014165174A3 publication Critical patent/WO2014165174A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P5/00—Preparation of hydrocarbons or halogenated hydrocarbons
    • C12P5/007—Preparation of hydrocarbons or halogenated hydrocarbons containing one or more isoprene units, i.e. terpenes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/14—Fungi; Culture media therefor
    • C12N1/145—Fungal isolates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/645—Fungi ; Processes using fungi
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
    • Y02E50/00—Technologies for the production of fuel of non-fossil origin
    • Y02E50/10—Biofuels, e.g. bio-diesel

Definitions

  • VOCs volatile organic compounds
  • 1,8-cineole commonly referred to as eucalyptol
  • eucalyptol is the pharmaceutically active component of eucalyptus oil, comprising 70-85% of the essential oil.
  • Traditional uses of eucalyptus oil primarily involve non-prescription pharmaceuticals, fragrances and degreasing detergents (Opdyke, 1975, Food and Cosmetics Toxicology 13 : 91-1 12; Hong and Shellock, 1991, American Journal of Physical Medicine and Rehabilitation 70:29-33; Leung, Y.
  • 1,8-Cineole also has potential applications in alternative fuel production as it has been shown to prevent phase separation when used as an additive in ethanol-gasoline fuel blends (Barton and Tjandra, 1989, Fuel 68: 1 1-17), and alternative fuels comprised of a gasoline/eucalyptus oil mixture (with 1,8-cineole as the major fuel component) resulted in an improved octane number and reduced carbon monoxide exhaust (U.S. Patent No. 4,297, 109).
  • fenchocamphorone is a derivative of fenchol via a fenchene intermediate, both of which are monoterpenes (Croteau, et al., 1988, Journal of Biological Chemistry
  • Fenchone also a monoterpene of similar derivations, is a volatile compound that is found as a major constituent of fennel seed oil (Azeez, S. (2008). Fennel. In Chemistry of Spices, pp. 227-241. Edited by Parthasarathy, V.A., Chempakam, B., &
  • Fennel oil is also considered an essential plant oil and is valued for its strong flavor, but is also recognized as an antioxidant, hepatoprotective agent, anticancer agent, and other biological activities have been described for it (Azeez 2008; Cosimi et al, 2009, Journal of Stored Products Research 45: 125-132).
  • 1,4-cyclohexadiene is a highly flammable cycloalkene that yields the natural monoterpene derivative, ⁇ -terpinene, a component associated with many essential oils.
  • 1,4- Cyclohexadiene also readily oxidizes to benzene by a number of different methods (Breton, et al, 2005, Electrochemistry Communications 7: 1445-1448; Smith and Gray, 1990, Catalysis Letters 6: 195-200; Hepworth et al, 2002, Aromatic Chemistry, pp. 129-134; Brooks, B.T. (1922).
  • the Cyclic Non-benzoid Hydrocarbons The Cyclohexane Series.
  • Benzene is a natural component of crude oil and gasoline and is a widely used chemical in the production of plastics, nylon, and resins, as well as some types of rubbers, detergents, lubricants, dyes, and pesticides (Agency for Toxic Substances and Disease Registry (ATSDR) (2007). Toxicological Profile for Benzene (Update). Atlanta, GA: U.S. Department of Public Health and Human Services, Public Health Service).
  • microorganisms can be a production source of chemical compounds, enzymes and other complexes that have industrial utility.
  • endophytes produce novel bioactive products stems from the idea that some endophytes may have coevolved with their respective higher plant, and as a result may produce certain phytochemicals characteristic of their hosts (Strobel and Daisy, 2003, Microbiology and Molecular Biology Reviews 67:491-502; Tan and Zou, 2001, Nat. Prod. Rep. 18:448-459).
  • the enormous diversity generated by the presence of microbial life forms is amplified by their ability to inhabit novel niches, ranging from deep ocean sediments to the earth's thermal pools.
  • Endophytic fungi inhabit one such biological niche and are characterized by their ability to asymptomatically colonize living plant tissues.
  • endophytes constitute a significant proportion (Smith, et al, 2008, PloS 1 3(8):e3052).
  • Ecosystems exhibiting the greatest plant diversity also seemingly exhibit the greatest abundance and diversity of microbial endophytes.
  • biological diversity implies chemical diversity as constant chemical innovation is required in such highly competitive ecosystems.
  • the search for novel endophytic microbes is ongoing, with activity of their natural products encompassing their use as antibiotics, antiviral compounds, anticancer agents, antioxidants, insecticides, antidiabetic agents, and
  • Hypoxylon spp.,whch is a fungal endophyte of Persea indica, an evergreen tree native to the Canary Islands, where it grows not in abundance but is found on several islands including Tenerife in the Laurisilva.
  • Persea spp. are also native to Central and South America and were later introduced into Southern California (Zentmyer, et al, 1990, California Avocado Society 1990 Yearbook 74:239-242).
  • the present invention relates to an isolated fungus that has the imperfect stage of Nodulisporium and produces at least one compound selected from the group consisting of 1, 8-cineole and 1 -methyl- 1, 4-cyclohexadiene.
  • the fungus is from the genus Nodulisporium.
  • the fungus is from the genus Hypoxylon.
  • the fungus is from the genus Annulohypoxylon.
  • another compound selected from the group consisting of 1, 8-cineole and 1 -methyl- 1, 4-cyclohexadiene is from the group consisting of 1, 8-cineole and 1 -methyl- 1, 4-cyclohexadiene.
  • the fungus is from the genus Nodulisporium.
  • the fungus is from the genus Hypoxylon.
  • the fungus is from the genus Annulohypoxylon.
  • the fungus is from the genus Daldinia. In another embodiment, the fungus is from the genus Xylaria. In another embodiment, the fungus is serially propagated. In another embodiment, the fungus is grown on or in a high-starch substrate. In another embodiment, the fungus is grown in a liquid medium. In another embodiment, fungus is grown on a solid medium. In another embodiment, the at least one compound is isolated from the culturing media. In another embodiment, the at least one compound is isolated from a vapor produced within a culturing container containing the culturing media.
  • the present invention also relates to an isolated fungus that has the imperfect stage of Nodulisporium and produces an alcohol.
  • the alcohol is ethanol.
  • the fungus is serially propagated.
  • the fungus is grown on or in a high-starch substrate.
  • the fungus is grown in a liquid medium.
  • the fungus is grown on a solid medium.
  • the at least one compound is isolated from the culturing media.
  • the at least one compound is isolated from a vapor produced within a culturing container containing the culturing media.
  • the present invention also relates to a method for producing at least one compound selected from the group consisting of 1, 8-cineole and 1 -methyl- 1,4- cyclohexadiene.
  • the method includes the step of culturing a microorganism on or within a culturing media in a container under conditions sufficient for producing the at least one compound.
  • the microorganism is an endophyte.
  • the endophyte is a fungus.
  • the fungus has the imperfect stage of Nodulisporium.
  • the fungus is from the genus Nodulisporium.
  • the fungus is from the genus Hypoxylon.
  • the fungus is from the genus Annulohypoxylon.
  • the fungus is from the genus Daldinia.
  • the fungus is from the genus Xylaria.
  • the microorganism is serially propagated.
  • the microorganism is grown on or in a high-starch substrate.
  • the microorganism is grown in a liquid medium.
  • the microorganism is grown on a solid medium.
  • the method further includes the step of isolating the at least one compound from the culturing media.
  • the method further includes the step of isolating the at least one compound from a vapor produced within the container.
  • the present invention also relates to a method for producing an alcohol.
  • the method includes the step of culturing a fungus having the imperfect stage of Nodulisporium on or within a culturing media in a container under conditions sufficient for producing the alcohol.
  • the alcohol is ethanol.
  • the fungus is serially propagated.
  • the fungus is grown on or in a high-starch substrate.
  • the fungus is grown in a liquid medium.
  • the fungus is grown on a solid medium.
  • Figure 1 depicts a PTR-mass spectrometer used to monitor VOC production by Hypoxylon sp.
  • the Hypoxylon sp. culture produced 100.5 mg dry weight of surface mycelium covering the 121.6 cm 2 agar slant at 7 days. Monitoring began 2.5 days after the fungus was inoculated onto the agar surface.
  • the inset shows the details of the hardware used to regulate gas flow into the culture flask.
  • the controller switch continuously changes input of gases from the control bottle (only PDA) to the fungal culture.
  • the computer screen shows the contiuous output of individual ions found in the gas phase.
  • Figure 2 comprising Figures 2A and 2B, depicts a 10-day old culture of Hypoxylon sp. grown on PDA from both the top side (2A) and botton side (2B). The darker aspect of the photos represents varying degrees of a greenish-tan coloration.
  • Figure 3 is an SEM image of a branched conidiophore Nodulisporium sp. (CI-4) depicting conidia and scars from the budding verticles of the conidiophore.
  • Figure 5 is a structural depiction of the fungal volatile organic compounds 1-methyl- 1,4-cyclohexadiene (top left), 1,8-cineole (top right), and (+)-a-methylene-a- fenchocamphorone (bottom).
  • Figure 6 is a PTR mass spectrum of the head space of a 5-day old culture of
  • Figure 7 is a graph of the production of individual compounds in the VOCs of Hypoxylon sp. as a function of time as measured and calculated from PTR mass spectral data.
  • the m/z at 121 is likely the series of protonated cyclic alkanes/alkenes whose mass is 120 (See Table 3 herein).
  • the terpenes including 1,8 cineole were calculated from contributions of compounds yielding masses 81, 137 and 155. All calculations are minus the PDA background control flask.
  • Figure 8 is a photograph of The Paleobiosphere as designed and built and used in the experiments described elsewhere herein.
  • Figure 9 is a series of photographs of paleobotanical examples of the plants used in the PBS experiments.
  • Figure 9A is an illustration oiPopulus sp.
  • Figure 9B is an illustration of Platanus sp.
  • Figure 9C is an illustration of Acer sp. .These specimens are known from late Cretaceous shales of the Western USA (the bars 1.5 cm).
  • Figure 9D is a photograh of petrified fungal hyphae penetrating petrified wood samples recovered from the oil rich Melstone, Mt. area.
  • Figure 9E is a photograh of petrified fungal hyphae penetrating petrified wood samples recovered from the oil rich Melstone, Mt. area.
  • Figure 10 is an illustration of the experiment set-up of the Paleobiosphere with a bed layer overlaid with a mixture of 200 g of shale, 25 g of plant materials and 10 g of 1 month old plant matter infested with the fungus.
  • the Trap Shale 200 g is contained in an envelope of stainless steel overlaying the shale/plant mixture.
  • On top is a lightly distributed fungal inoculum of infested plant material.
  • Figure 1 is a series of photographs representing the PBS (under the "Trap Shale” layer) at the termination of the experiment.
  • Figure 11A is a photograph illustrating how the leaves and shale materials are completely covered with fungal hyphae.
  • Figure 1 IB is a photograph illustrating how the "Trap Shale' ' itself (when the stainless steel mesh screen was removed) was damp and covered with fungal hyphae.
  • FIG. 11C is a photograph of an SEM image of fungal hyphae growing on the surface of a shale particle (treatment shale).
  • Figure 1 ID is a photograph of a SEM image illustrating no evidence of any hyphae on the control shale.
  • Figure 12 is a series of photographs representing the morphological characteristics of 25-2A.
  • Figure 12A is a photograph of Thelypteris angustifolia or Broad Leaf Maiden Fern as the host of the endophytic fungus Nodulisporium sp. ( ⁇ 25-2 ⁇ ). The bar equals 1 cm.
  • Figure 12B is a SEM image of the conidiophore of Nodulisporium sp. showing a clump of singly borne conidiophores. The bar equals 2 ⁇ .
  • Figure 13 is an illustration of the phylogenetic analysis of ⁇ 25-2 ⁇ .
  • Figure 13A is a graph showing how the evolutionary history of 25-2A
  • FIG. 12B is a table of the distance matrix depicting the percent sequence identity and divergence of the 18S-ITS-5.8S ribosomal gene sequence of NI25-2A with its close relatives, constructed by the MegAlign module of the DNASTAR (Lasergene) software.
  • Figure 14 is a photograph of the Nodulisporium sp. or imperfect stage of EC-12 (Nodulisporium sp.) as seen by SEM. Note the conidiophores bearing numerous conidia.
  • Figure 15 is a phylogenetic tree, depicting the evolutionary position of the endophyte EC12, constructed using the UPGMA method (Griffin et al, 2010, Microbiology 156:3814- 3829). The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (500 replicates) is shown next to the branches (Strobel, 2006, Curr. Opin. Microbiol. 9:240-244). The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Maximum Composite Likelihood method as previously described (Tamura et al, 2007, Mol. Biol. Evol. 24: 1596-1599) and are in the units of the number of base substitutions per site. All positions containing gaps and missing data were eliminated from the dataset (Complete deletion option).
  • Figure 16 is a SEM image of Nodulisporium sp. isolate Ti-13, which was isolated from Cassia sp. in the hills of western Thailand. Under SEM, Ti-13 looks identical to a Nodulisporium sp.
  • Figure 17 is an illustration of Nodulisporium sp. isolate Ti-13. The spores are 5-6 microns x 2.5 "4 microns.
  • Figure 18 is a dendogram of the Ti-13.
  • the numbers designate the support branch values, confirming that Ti-13 is a Nodulisporium sp.
  • the present invention relates to isolated fungal lines capable of producing an impressive spectrum of volatile organic compounds (VOCs), most notably 1, 8-cineole, 1- methyl-1, 4-cyclohexadiene, and (+)-a-methylene-a-fenchocamphorone, among many others (see Table 3, below).
  • VOCs volatile organic compounds
  • the present invention also relates to methods of producing such VOCs from microorganisms, and collecting or recovering the produced VOCs for commercial and/or industrial use.
  • the microorganism is a fungus.
  • the present invention is based on the discovery that selected fungi, including numerous Hypoxylon spp., produce an impressive spectrum of VOCs, most notably 1, 8- cineole, 1 -methyl- 1, 4-cyclohexadiene, and (+)-a-methylene-a-fenchocamphorone.
  • Media containing starch and/or sugar related substrates best supports VOC production by fungus.
  • Direct on-line quantification of VOCs was measured by proton transfer mass spectrometry (PTR-MS) covering a continuous range, with optimum VOC production occurring at 6 days at 145 ppmv with a rate of production of 7.65 ppmv/hr.
  • 1, 8-cineole (a monoterpene) is produced by a microorganism, which represents a novel and important source of this compound.
  • 1, 8-cineole is an octane derivative and has potential use as a fuel additive, as do the other VOCs of this organism, listed in Table 3, below.
  • fungal sourcing of this compound and other VOCs as produced by Hypoxylon sp. and other fungi described herein greatly expands their potential applications in medicine, industry, and energy production.
  • an element means one element or more than one element.
  • “About” as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ⁇ 20% or ⁇ 10%, more preferably ⁇ 5%, even more preferably ⁇ 1%, and still more preferably ⁇ 0.1% from the specified value, as such variations are appropriate to perform the disclosed methods.
  • hydrocarbon generally refers to a chemical compound that consists of the elements carbon (C) and hydrogen (H). All hydrocarbons consist of a carbon backbone and atoms of hydrogen attached to that backbone. Hydrocarbons are of prime economic importance because they encompass the constituents of the major fossil fuels (coal, petroleum, natural gas, etc.) and biofuels, as well as plastics, waxes, solvents and oils.
  • fungus or "fungi” includes a wide variety of nucleated, spore-bearing organisms that are devoid of chlorophyll. Examples of fungi include yeasts, molds, mildews, rusts, and mushrooms.
  • bacteria includes any prokaryotic organism that does not have a distinct nucleus.
  • isolated means altered or removed from the natural state or biological niche through the actions of a human being.
  • antibiotic includes any substance that is able to kill or inhibit a microorganism. Antibiotics may be produced by a microorganism or by a synthetic process or semisynthetic process. The term, therefore, includes a substance that inhibits or kills fungi for example, cycloheximide or nystatin.
  • culturing refers to the propagation of organisms on or in solid or liquid media of various kinds.
  • an effective amount is an amount sufficient to effect beneficial or desired results.
  • An effective amount can be administered in one or more administrations.
  • an “effective amount” is that amount sufficient to ameliorate, stabilize, reverse, slow or delay progression of the target infection or disease states.
  • metabolite or “volatile” refers to any compound, substance or byproduct of a fermentation of a microorganism that has a biological activity.
  • mutant refers to a variant of the parental strain as well as methods for obtaining a mutant or variant in which the desired biological activity is similar to that expressed by the parental strain.
  • the "parent strain” is defined herein as the original fungus (e.g. Hypoxylon) strains before mutagenesis. Mutants occur in nature without the intervention of man. They also are obtainable by treatment with or by a variety of methods and compositions understood by those of skill in the art. For example, parental strains may be treated with a chemical such as N-methyl-N'-nitro-N-nitrosoguanidine, ethylmethanesulfone, or by irradiation using gamma, x-ray, or UV-irradiation, or by other means.
  • a chemical such as N-methyl-N'-nitro-N-nitrosoguanidine, ethylmethanesulfone, or by irradiation using gamma, x-ray, or UV-irradiation, or by
  • variant refers to a strain having all the identifying characteristics of the strains of fungus and can be identified as having a genome that hybridizes under conditions of high stringency to the genome of the organism.
  • a variant may also be defined as a strain having a genomic sequence that is greater than 85%, more preferably greater than 90% or more preferably greater than 95% sequence identity to the genome of the organism.
  • a polynucleotide or polynucleotide region (or a polypeptide or polypeptide region) has a certain percentage (for example, 80%, 85%, 90%, or 95%) of "sequence identity" to another sequence, which means that, when aligned, that percentage of bases (or amino acids) are the same in comparing the two sequences.
  • range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, 6 and any whole and partial increments therebetween. This applies regardless of the breadth of the range.
  • a search for endophytes hosted by the evergreen tree Persea indica revealed the presence of a fungal sp. having the imperfect stage of Nodulisporium, as described herein.
  • An examination of this organism revealed that it produces important VOCs including, without limitation, 1, 8-cineole; 1 -methyl- 1, 4-cyclohexadiene, and (+)-a-methylene-a- fenchocamphorone (see Table 3, below).
  • These compounds have potential industrial utility, such as fuels or additives as per the VOCs of some other endophytic fungi now known as MycodieselTM (Strobel, et al, 2008, Microbiology 154:3319-3328).
  • the present invention includes an isolated fungus capable of producing at least one VOC.
  • an isolated fungus capable of producing at least one VOC.
  • the following fungal isolates having the imperfect stage of Nodulisporium, each being capable of producing at least one VOC were deposited under the terms of the Budapest Treaty with the ARS Culture Collection (NRRL), 1815 North University Street, Peoria, Illinois 61604-3999 USA, on May 1 1, 201 1 and assigned the corresponding Accession Numbers:
  • the present invention provides methods of producing VOCs from microorganisms, and collecting or recovering the produced VOCs for commercial and/or industrial use.
  • any one of the fungi described herein can produce an impressive spectrum of volatile organic compounds (see Tables 3, 6, 10-1 1, and 15-17, below) including, without limitation, 1,8-cineole, 1 -methyl- 1 ,4-cyclohexadiene, (+)-a- methylene-a-fenchocamphorone, the structures of which are depicted in Figure 5. It should be appreciated that the present invention is not limited to production of the aforementioned VOCs by Hypoxylon or any other fungus having the imperfect stage of Nodulisporium.
  • the present invention includes production of VOCs, particularly 1,8-cineole, 1- methyl-l,4-cyclohexadiene, and (+)-a-methylene-a-fenchocamphorone by any fungus, or for that matter, any microorganism.
  • VOCs particularly 1,8-cineole, 1- methyl-l,4-cyclohexadiene, and (+)-a-methylene-a-fenchocamphorone by any fungus, or for that matter, any microorganism.
  • 1,8-cineole can also be produced by an isolated Nodulisporium sp., such as PC-37-24, and is therefore also contemplated as forming part of the present invention.
  • the present invention relates to endophytic fungi that produce volatile organic compounds, such as hydrocarbons, from isolates of Nodulisporium spp, Hypoxylon spp., Daldinia spp., Xylaria spp., and Annulohypoxylon spp.
  • volatile organic compounds such as hydrocarbons
  • This novel, renewable source of hydrocarbons is desirable because it provides a supplement to the existing limited resources of non-renewable hydrocarbons.
  • Hypoxylon isolates can also be classified as an endophytic Nodulisporium sp. or Daldinia sp.or Annulohypoxylon sp., depending on the fungal identification methodology used.
  • almost all fungi have a perfect (sexual stage) and an imperfect stage (non sexual), and each is given a name.
  • Nodulisporium-like organisms can have the perfect stage of Hypoxylon, Daldinia, Annulohypoxylon, or Xylaria. Therefore, as contemplated herein, fungi identified as any one of Nodulisporium spp., Hypoxylon spp., Daldinia spp., Xylaria spp., or
  • Annulohypoxylon spp. form part of the present invention for the generation of VOCs, as described herein.
  • strains Co27-5, CI-4A, Ti-13, Ec-38, ⁇ 25-2 ⁇ , Th-9, and Fl-9 are generally classified as Hypoxylon, they each have the imperfect stage of Nodulisporium sp.
  • strain EC- 12 is generally classified as Daldinia, it has the imperfect stage of Nodulisporium sp.
  • strain D-6 is generally classified as Annulohypoxylon, it has the imperfect stage of Nodulisporium sp.
  • the fungi of the present invention include all anamorphs and teleomorphs, to the extent such forms exist and are available.
  • the Hypoxylon strains Co27-5, CI-4A, Ti-13, Ec-38, 25-2A, Th-9, and Fl-9 have Nodulisporium sp. as their anamorphic stage.
  • the difference between an anamorph and teleomorph is that one is the asexual state and the other is the sexual state, where the two states exhibit different morphology under certain conditions.
  • these fungi may have two names.
  • the teleomorph name describes the fungus when reproducing sexually
  • the anamorph name refers to the fungus when reproducing asexually.
  • the holomorph name refers to the "whole fungus"
  • microorganisms such asNodulisporium spp., Hypoxylon spp., Dcildinici spp., .Xylaria spp., Annulohypoxylon spp., can be used in combination with other microbes (e.g. yeasts or other bacteria) for the large scale production of biofuels.
  • other microbes e.g. yeasts or other bacteria
  • the present invention also includes isolated strains of a
  • Nodulisporium, Hypoxylon, Daldinia, Xylaria, or Annulohypoxylon wherein the isolated fungal strain was serially propagated.
  • some of the characteristics of the strain may change. Such changes include deletion or suppression of metabolic pathways, an increase in certain metabolic pathways, changes to the chromosome, genes and/or operons (e.g. via mutations or changes in the regulatory factors that control the expression level of said genes or operons).
  • a strain of Hypoxylon may have changes in its metabolic characteristic and/or genetic make-up as compared to Hypoxylon isolates Co27-5, CI-4A, Ti-13, Ec-38, Ni-25 2A, Th-9, or Fl-9.
  • Such changes to the metabolic characteristics and/or genetic make-up may increase and/or decrease the production of the specific compounds listed in Table 3.
  • the present invention also provides a method for producing volatile organic compounds, such as hydrocarbons.
  • the method comprises culturing isolates of Nodulisporium spp, Hypoxylon spp., Daldinia spp., Xylaria spp., Annulohypoxylon spp., and under conditions sufficient for producing VOCs, and collecting or recovering the produced VOCs.
  • the methods of the present invention also include any combination of procedures and steps used in the culturing of fungi and recovery of at least one VOC, as described hereinthroughout.
  • the present invention relates to endophytic fungi that produce volatile organic compounds, such as the hydrocarbons listed in Tables 3, 6, 10-11, and 15-17, below.
  • volatile organic compounds such as the hydrocarbons listed in Tables 3, 6, 10-11, and 15-17, below.
  • 1,8-cineole, 1 -methyl- 1 ,4-cyclohexadiene, and (+)-a-methylene-a-fenchocamphorone the structures of which are depicted in Figure 5.
  • Each of these compounds is either itself a monoterpene or is a direct derivative of a monoterpenic compound.
  • 1,8-cineole is not known to be a constituent of essential oils collected from leaves of a Persea indica plant in California (Weyerstahl, et al, 1993, Flavour and Fragrance Journal 8:201-207). However, this possibility should not be disregarded, given the highly diverse environment of this isolate.
  • Hypoxylon sp. to synthesize monoterpenic compounds typically associated with antimicrobial activity exemplifies the ability for microorganisms to inhabit essential oil producing plants, and their potential role in acquiring the biosynthetic pathways of these compounds should not be overlooked (Table 2).
  • 1, 8 Cineole has a broad spectrum of uses, from over-the-counter medical ointment to solvent/degreasers to flavoring/fragrances to alternative fuel.
  • production of 1,8-cineole by a fungal isolate is significant and greatly expands its potential for a broad spectrum of industrial applications.
  • the VOCs may be hydrocarbons, and may be useful for the production of biofuels, plastics, plasticizers, antibiotics, rubber, fuel additives, and/or adhesives.
  • hydrocarbons can also be used for electrical power generation and heating.
  • the chemical, petrochemical, plastics and rubber industries are also dependent upon hydrocarbons as raw materials for their products.
  • biofuel refers generally to any fuel that derives from biomass, i.e. recently living organisms or their metabolic byproducts, such as manure from cows, or a hydrocarbon produced by fungi.
  • a biofuel may be further defined as a fuel derived from a metabolic product of a living organism.
  • the present invention represents the first time that 1,8-cineole and the other volatile products listed in Table 3 can be produced by endophytic fungi. Prior to this, the only known biological source of 1,8-cineole was from plant tissue. Production of VOCs from fungi represents a far superior commercial production model than from plants.
  • Biosynthesis of 1,8-cineole involves its conversion from geranyl pyrophosphate by 1,8-cineole cyclase (cineole synthase), whose activity is inhibited by cysteine- and histidine- directed reagents but protected by substrate-metal ion complexes, with the ether oxygen atom of this oxygen-containing terpene being solely derived from water (Croteau, et al, 1994,
  • fenchocamphorone is also converted from geranyl pyrophosphate and proceeds through the pathway as the intermediate (-)-(3R)- linalyl pyrophosphate via (-)-ewifo-fenchol cyclase (synthase) which subsequently cyclizes in the presence of the (4R)-a-terpinyl and (lR,5R)-pinyl cations to form (-)-endo-Fenchol which can further oxidize to ⁇ / ⁇ -fenchocamphorone (Croteau, et al., 1988, Journal of
  • Ethanol is an important compound for the fuel industry, as it is a component of biofuel and can be used as an additive in gasoline. As depicted in Figures 6 and 7, ethanol was identified as a VOC isolated from the headspace of a microorganism of the present invention. Ethanol may be produced and isolated using any method described hereinthroughout.
  • any substrate suitable for promoting fungal growth may be used in the production of VOCs, including without limitation any of the components listed in Table 4, in any ratios and combinations, as would be understood by those skilled in the art.
  • high starch substrates promote optimal VOC production, as demonstrated by substrate utilization assays containing high amounts of starch as a carbohydrate source (Table 4).
  • cellulose may also be a suitable substrate. Given the enormous volumes of accumulating cellulitic biomass and the utilization of foodstuff grains in alcohol (fuel) production, microorganisms that utilize cellulose for the production of VOCs are quite attractive.
  • the culture media for culturing fungi may include substrates comprising oatmeal, barley, or potato agar bases.
  • the culture media may also be a PDA medium, a cellulose medium, and may include starch, glucose, or any combination of components listed in Table 4.
  • the selected fungal strain may be grown in a medium containing any combination of inorganic salts, organic nitrogen sources, such as peptones, defatted cotton seed flour, corn steep liquor, or yeast extract and carbon source.
  • examples of carbon source may include, but is not limited to, glucose, lactose, sucrose, cellulose or other carbohydrates.
  • the present invention should not be limited by the type or amount of growth media used, and should include use of any media suitable for cultivating fungi as would be understood by those skilled in the art.
  • these conditions can also include culturing fungi in the absence of oxygen (anaerobic conditions) or in reduced oxygen conditions (e.g., microaerophilic conditions).
  • the isolated fungi of the present invention can be cultured using standard methods as would be understood by those skilled in the art.
  • the fungal cultures can be cultured on a large scale for commercial use, by using conventional fermentation techniques.
  • fermentation is used broadly to refer to any controlled fungal culturing conditions.
  • an inoculum of said growth culture Prior to large scale growth an inoculum of said growth culture is generally cultured.
  • the fungi can be cultured in a bioreactor vessel for a scaled up production of VOCs.
  • Any conventional bioreactor vessel can be used as the vessel for the purpose of this invention.
  • the vessel may be made of materials such as stainless steel, glass, plastic, and/or ceramics, and may have a volume of from about 100 ml to 10,000 L or larger.
  • the bioreactor vessel may be connected to a series of storage flasks that contain nutrient solutions and solutions for maintaining and controlling various parameters of the cultivation and VOC recovery process.
  • storage flasks may contain nutrient solutions and solutions for maintaining and controlling various parameters of the cultivation and VOC recovery process.
  • there may be separate storage flasks for individual supply of substrates to the vessel, which substrates serve as the carbon, nitrogen or mineral source for the living cells in the vessel.
  • Fed Batch culture is a variation on ordinary batch culture and involves the addition of a nutrient feed to the batch. Cells are cultured in a medium in a fixed volume. Before the maximum cell concentration is reached, specific supplementary nutrients are added to the culture. The volume of the feed is minimal compared to the volume of the culture.
  • Fed batch culture typically proceeds in a substantially fixed volume, for a fixed duration, and with a single harvest either when the cells have died or at an earlier, predetermined point.
  • the cells are initially grown in a fixed volume of medium.
  • fresh medium is pumped into the bioreactor before maximum cell concentration is reached.
  • the spent media containing a proportion of the cells, is continuously removed from the bioreactor to maintain a constant volume.
  • the process also removes the desired product, which can be continuously harvested, and provides a continuous supply of nutrients, which allows the cells to be maintained in an exponentially growing state.
  • the process can be operated indefinitely.
  • Continuous culture is characterized by a continuous increase in culture volume, an increase and dilution of the desired product, and continuous maintenance of an exponentially growing culture.
  • Perfusion culture is similar to continuous culture except that, when the medium is pumped out of the reactor, cells are not removed. As with a continuous culture, perfusion culture is an increasing-volume system with continuous harvest that theoretically can continue indefinitely. Recovery of VOCs
  • VOCs listed in Table 3 can be produced by the selected fungi isolate.
  • common separation techniques can be used to remove the cells from the broth or agar
  • common isolation procedures such as (without limitation) extraction, distillation, and carbocolumn trap procedures, can be used to obtain VOCs from the cell-free broth or agar. See, for example, U.S. Pat. Nos. 4,275,234, 5,510,526; 5,641,406, and 5,831, 122, and International Patent Application Number WO 93/00440, each of which is hereby incorporated by reference in its entirety.
  • Fractional distillation and/or absorption chromatography are also non-limiting examples of methods to extract the desired product produced by fungal isolates of the present invention.
  • Fractional distillation is the separation of a mixture into its component parts, or fractions, such as in separating chemical compounds by their boiling point by heating them to a temperature at which several fractions of the compound will evaporate.
  • Absorption chromatography is a physical separation method in which the components of a mixture are separated by differences in their distribution between two phases, one of which is stationary (stationary phase) while the other (the mobile phase) moves through it in a definite direction. The substances must interact with the stationary phase to be retained and separated by it.
  • Gas chromatography is a well known technique for fractionating and determining the relative amounts of various components in a sample containing a mixture of compounds of differing volatilities. For example, the sample is vaporized and the entire resulting quantity of gases is passed through an analytical chromatography column. Chromatographic processes such as gas chromatography can rapidly determine the volatiles content of a multicomponent sample, such as would be produced by the fungal isolates of the present invention.
  • Pressure Swing Adsorption may be used to separate some gas species from a mixture of gases under pressure according to the species' molecular characteristics and affinity for an adsorbent material. It operates at near-ambient temperatures and so differs from cryogenic distillation techniques of gas separation. Special adsorptive materials (e.g., zeolites) are used as a molecular sieve, preferentially adsorbing the target gas species at high pressure. The process then swings to low pressure to desorb the adsorbent material.
  • Special adsorptive materials e.g., zeolites
  • a carbotrap column may be used for the trapping and recovery of VOCs.
  • VOCs produced by a fungal culture pass through a trapping column containing adsorption material, and are trapped within the column as they are captured by the adsorption material. The VOCs may then be released from the trapping column by simultaneously heating the column while purging with a gas, and collecting the VOCs in a cold trap condenser.
  • the present invention also includes mutant or engineered fungi that ultimately increase the production yield of at least one VOC, or the speed at which the mutant or engineered fungi can produce at least one VOC.
  • Mutant or engineered fungi are obtainable by treatment of fungi with or by a variety of methods and compositions understood by those of skill in the art.
  • parental strains may be treated with a chemical such as N- methyl-N'-nitro-N-nitrosoguanidine, ethylmethanesulfone, or by irradiation using gamma, x- ray, or UV-irradiation, or by other means.
  • the present invention also includes identifying and cloning genes that encode for production of at least one VOC from the genomes of each fungus described herein.
  • the Hypoxylon genome is probed for the gene or genes (e.g. an operon) that encode the synthetic pathways that produce a VOC from Table 3, such as 1,8-cineole, 1 -methyl- 1,4-cyclohexadiene, and (+)-a-methylene-a- fenchocamphorone.
  • the present invention encompasses an isolated nucleic acid molecule from fungi encoding a polypeptide involved in the synthesis or production of at least one VOC.
  • an isolated nucleic acid molecule is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to said isolated nucleic acid molecule from any one of the fungi isolates described herein.
  • a polypeptide sequence is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a polypeptide from any one of the fungi isolates described herein.
  • Methods to clone and/or probe genomes for synthetic pathways may include creating cDNA and/or genomic libraries, and screening the libraries for genes that produce the VOC synthetic pathways.
  • the present invention comprises a DNA and/or chromosomal library of any one of the fungi isolates described herein.
  • the library is cloned into a vector that can replicate in a prokaryotic cell and/or eukaryotic cell.
  • the eukaryotic cell is a fungal cell.
  • the library is a lambda phage, Yeast Artificial Chromosome, Bacterial Artificial Chromosome, and/or cDNA.
  • the library is screened for production of VOCs from Table 3, such as 1,8-cineole, 1 -methyl- 1,4-cyclohexadiene, and (+)-a-methylene-a- fenchocamphorone.
  • Another method for determining the gene, genes and/or operon(s) that encode for the production of VOCs include mutagenizing the genome of any one of the fungi described herein and looking for an increase, addition, reduction or removal of a specific VOC. This can be accomplished via chemical and/or transposon mutagenesis. Once a gene, genes and/or operon(s) is identified, said gene, genes or operon(s) can be cloned and/or isolated.
  • one embodiment of the invention comprises an isolated nucleic acid of any one of the fungi described herein, wherein the nucleic acid molecule is cloned into a vector.
  • said nucleic acid molecule encodes for a gene, genes, or operon(s) that encode for proteins involved in the production of VOCs of Table 3.
  • the vector autonomously replicates or integrates into the host's chromosome.
  • said vector is transformed or transfected into a heterologous cell.
  • said heterologous cell is selected from the group consisting of a prokaryotic or eukaryotic cell.
  • the present invention also encompasses variants and fragments of polynucleotides and/or proteins of any one of the fungi described herein that produce or are part of the pathway(s) that produce VOCs.
  • the variants may contain alterations in the nucleotide and/or amino acid sequences of the constituent proteins.
  • the term "variant" with respect to a polypeptide refers to an amino acid sequence that is altered by one or more amino acids with respect to a reference sequence.
  • the variant can have "conservative" changes, or
  • nonconservative changes e.g., analogous minor variations can also include amino acid deletions or insertions, or both.
  • nucleotides can be sequenced to ensure that the correct coding regions were cloned and do not contain any unwanted mutations.
  • Nucleic acid molecules encoding one or more biosynthetic enzyme or protein, and orthologs and homologs of these sequences, may be incorporated into transformation or expression vectors of any one of the fungi described herein.
  • the term "vector” refers generally to a nucleic acid molecule as introduced into a host cell, thereby producing a transformed host cell.
  • a vector may include nucleic acid sequences that permit it to replicate in a host cell, such as an origin of replication.
  • a vector may also include one or more selectable marker genes and other genetic elements known in the art.
  • a transformed cell is a cell into which has been introduced a nucleic acid molecule by molecular biology techniques. The term encompasses all techniques by which a nucleic acid molecule might be introduced into such a cell, including transfection with viral vectors, transformation with plasmid vectors, and introduction of naked DNA by electroporation, lipofection, and particle gun acceleration.
  • the heterologous organism can be grown to produce and purify the desired VOCs, including those listed in Table 3.
  • the present invention also includes a method for generating mutant strains of a fungus with an increased production rate or production amount of at least one compound, such as 1, 8-cineole, 1 -methyl- 1, 4-cyclohexadiene, and (+)-a-methylene-a- fenchocamphorone, or any other compound listed in Table 3, below.
  • the method includes the steps of mutating spores of the fungus, culturing the mutated spores, and screening the cultures of mutated spores for enhanced production rate or production amount of at least one compound selected from the group consisting of 1, 8-cineole, 1 -methyl- 1, 4-cyclohexadiene, and (+)-a-methylene-a-fenchocamphorone.
  • kits comprising one or more containers filled with one or more of the ingredients of the compositions of the invention.
  • the present invention provides kits that can be used in any of the methods described herein.
  • a kit comprises at least one Nodulisporium sp., Hypoxylon sp., Daldinia sp.,
  • kits Xylaria sp., Annulohypoxylon sp., or in one or more containers.
  • the organism can be supplied frozen in media, freeze dried and/or as spores.
  • the kit may also include instructional material for growing the fungi under optimal conditions for optimal VOC production.
  • the methods in the instructions may include specific bioreactor volumes, purification schemes, optimal temperature, pH, and/or other conditions.
  • the kit may also include the growth media.
  • the media contained in the containers of these kits may be present as a ready-to-use formulation, or as a more concentrated formulation.
  • the media can be supplied in dry powder.
  • a kit can comprise a dry power of the media of the invention and a liquid to suspend the media.
  • the liquid may be water or buffers known in the art. Filters for sterilization of the media may also be provided.
  • Example 1 Hypoxylon sp, an endophyte of Persea indica, producing 1, 8-cineole and other bioactive volatiles with fuel potential
  • Endophytic fungal culture, CI-4A was obtained as an endophyte from an evergreen tree (Persea indica), native to the Canary Islands.
  • Persea indica One small limb was excised from Persea indica found growing on the island of Tenerife, Spain, at - 28° 32' 23"; W- 16° 16' 16".
  • Other plant species sampled from this same island included Acacia sp., Pinus canadensis, Prunus lusitanica and Rhamnus glandifolia, none of which fostered recovery of CI-4.
  • the fungus was stored by placing small plugs of PDA supporting mycelial growth in 15% glycerol at -70°C.
  • An alternative storage method was also utilized in which the fungus colonized sterile barley seed, which was subsequently air dried and then stored at -70°C.
  • SEM Scanning electron microscopy
  • the fungus was grown on PD broth for 7 days, after which the mycelium was harvested and the genomic DNA extracted using DNeasy Plant and Fungi Mini Kit (Qiagen), according to the manufacturer's directions.
  • the internal transcribed spacer (ITS) regions of the fungus were amplified using PCR with the universal ITS primers ITS1 (5' TCC GTA GGT GAA CCT GCG G 3') (SEQ ID NO: 1) and ITS4 (5' TCC TCC GCT TAT TGA TAT GC 3') (SEQ ID NO:2). All other procedures were carried out as previously described by Ezra.
  • the DNA was sequenced and submitted to GenBank. Sequences obtained in this study were compared to the GenBank database using the BLAST software.
  • a phylogenetic tree was assembled using MEGA4 (Tamura, et al, 2007, Molecular Biology and Evolution 24: 1596-1599) and the Neighbor- Joining method (Saitou and Nei, 1987, Molecular Biology and Evolution 4:406-425) with positions containing gaps and missing data eliminated from the dataset (complete deletion option).
  • VOCs produced by CI-4A were tested for inhibitory antimicrobial activity against selected pathogenic fungi and bacteria according to a bioassay test system previously described for analysis of VOCs produced by Muscodor albus (Strobel, et al, 2001,
  • the assays were conducted by removing a 2.5 cm wide strip of agar from the mid- portion of a standard Petri plate of PDA, creating two isolated halves of agar.
  • the fungus (CI- 4) was inoculated onto one half-moon agar piece and incubated at 23 °C for six days to allow for optimum production of volatile compounds.
  • Test pathogens were inoculated onto the half-moon section of agar opposite the half-moon section inoculated with CI-4. The plate was then wrapped with a single piece of Parafilm and incubated at 23°C for 24 hours.
  • a variety of selected media was used to determine a combination of substrates that best facilitated VOC production by CI-4.
  • a single plug taken from an actively growing culture of CI-4A on PDA was used to inoculate each agar based medium.
  • Preliminary quantification of 1,8 cineole was estimated by a human olfactory method since this compound is readily sensed by smell.
  • yeast extract 0.1 g ⁇ 1 plus salts yeast extract 0.1 g ⁇ 1 plus salts
  • peptone 0.1 g ⁇ 1 plus salts C
  • D cellulose 25 g ⁇ 1 plus salts and peptone 0.1 g ⁇ 1
  • E starch 25 g ⁇ 1 plus salts and yeast extract 0.1 g ⁇ 1
  • F starch 25 g ⁇ 1 plus salts and peptone 0.1 g ⁇ 1
  • G glucose 25 g ⁇ 1 plus salts and yeast extract 0.1 g ⁇ 1
  • H glucose 25 g ⁇ 1 plus salts and peptone 0.1 g ⁇ 1
  • I cellobiose 25 g ⁇ 1 plus salts and yeast extract 0.1 g ⁇ ⁇ 1 ⁇
  • poly dimethyls iloxane on a Stable Flex fibre was placed through a small hole drilled in the side of the Petri plated and exposed to the vapour phase for only 5 min due to the high concentration of fungal VOCs.
  • the syringe was then inserted into the splitless injection port of a Hewlett Packard 6890 gas chromatograph containing a 30 m x 0.25 mm ID. ZB Wax capillary column with a film thickness of 0.50 ⁇ .
  • the column was temperature programmed as follows: 30 °C for 2 min increased to 220 °C at 5 °C min "1 .
  • the carrier gas was ultra high purity helium, and the initial column head pressure was 50 kPa.
  • the fiber Prior to trapping the volatiles, the fiber was conditioned at 240 °C for 20 min under a flow of helium gas. A 30 sec injection time was used to introduce the sample fiber into the GC.
  • the gas chromatograph was interfaced to a Hewlett Packard 5973 mass selective detector (mass spectrometer) operating at unit resolution. The MS was scanned at a rate of 2.5 scans per second over a mass range of 35-360 amu. Data acquisition and data processing were performed on the Hewlett Packard ChemStation software system. Tentative identification of the compounds produced by CI-4A was made via library comparison using the NIST database, and all chemical compounds described in this report use the NIST data base chemical terminology.
  • PTR-MS was used to quantify production of fungal volatiles on a continuous monitoring basis beginning with a 2.5 day old culture growing on a 300 ml slant of PDA in a 1L bottle at 20 ⁇ 2 °C.
  • the bottle possessed an O-ring sealed cap that had been modified to possess both inlet and outlet tubes with 10 std cc/min of purified compresed air (Ezra, et al, 2004, Plant Science 166: 1471-1477)(Fig. 1).
  • the PTR-MS instrument ionizes organic molecules in the gas phase through their reaction with ]3 ⁇ 40 + , forming mostly protonated molecules (MH + , where M is the neutral organic molecule) which can then be detected by a standard quadrupole mass spectrometer.
  • This process can be run on real air samples with or without dilution, since the primary constituents of air (nitrogen, oxygen, argon and carbon dioxide) have a proton affinity less than water and thus are not ionized.
  • Most organic molecules excepting alkanes have a proton affinity greater than water and are therefore ionized and detected.
  • PTR-MS is that from the known or calculated quantities, the reaction time, the amount of ]3 ⁇ 40 + present, and the theoretical reaction rate constant for the proton transfer reaction, the absolute concentration of constituents in a sample can be quantified (Lindinger, et al, 1998, Int J Mass Spectrom Ion Process 173 : 191-241).
  • an enormous advantage of PTR-MS is that it can be run in real time and continuously produce data on the concentrations of specific ions of interest.
  • the product ion distribution is determined from mixtures prepared from pure standards.
  • the degree of susceptibility of the assay test organisms was dependent upon the age of the Hypoxylon sp. culture to which they were exposed for 24 hr (Tablel).
  • Table 1 Progressive (time course) bioassay showing susceptibility of selected fungal pathogens to Hypoxylon sp. VOCs as a function of Hypoxylon sp. culture age with a 24 hr exposure to the fungal VOCs. The percentages reported are relative to growth of the test organism on a PDA plate minus Hypoxylon sp.
  • Rhizoctonia solani 75.0% ⁇ 35.3 75.0% ⁇ 35. .3 37.5% ⁇ 53.0 87.5% ⁇ 17 .6 100.0% ⁇ 0.0
  • Trichoderma viridae 16.6% ⁇ 16.8 4.7% ⁇ 6. .7 19.0% ⁇ 6.73 23.8% ⁇ 0. .0 4.7% ⁇ 0.0
  • a progressive (time course) assay using ten different fungal pathogens was designed to determine the time point at which maximum sensitivity of the test organisms occurred which may also relate to the maximum point of VOC production by the fungus. Inhibitory activity of VOCs produced after three, four, five, six, and seven days was compared and maximum inhibition, suggesting the highest concentration of volatile bioactive substances, occurred at six days with eight of the ten test organisms exhibiting maximum inhibition at this time point.
  • the most sensitive test organisms to the VOCs of Hypoxylon sp. were Phytophthora spp., Sclerotinia sclerotiorum, Aspergillus fumigatus, and Cercospora beticola (Table 1).
  • Inhibition values were calculated as a percentage of growth inhibition as compared to an untreated control test organism at a 3 day exposure. Tests were conducted in triplicate and results varied as indicated by standard deviations. All organisms were viable after exposure to fungal VOCs.
  • Botrytis cinerea 100.0% ⁇ 0.0 A Colletotrichum lagenarium 36.1% ⁇ 12.7 A
  • Rhizoctonia solani* 66.6% ⁇ 57.7 A
  • albus is yet to be matched by a VOC producing fungus (Strobel, et al, 2001, Microbiology 147:2943-2950).
  • the ability to withstand its own monoterpenic antimicrobials may or may not be linked to its ability to withstand the potent volatile antimicrobials produced by M. albus.
  • 1, 8-cineole (a monoterpene) is produced by a microorganism, which represents a novel and important source of this compound.
  • This monoterpene is an octane derivative and has potential use as a fuel additive as do the other VOCs of this organism.
  • fungal sourcing of this compound and other VOCs as produced by Hypoxylon sp. greatly expands their potential applications in medicine, industry, and energy production.
  • Example 2 The Paleobiosphere: a novel device for the in vivo testing of hydrocarbon production utilizing microorganisms
  • 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 D-6, an 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, l,2,3,5,6,7,8,8a-octahydro-l,4-dimethyl- 7- (1-methylethenyl), [lS-(la,7a,8ab)].
  • the instrument has been constructed from stainless steel parts including the main chamber and all valves, ports and traps. Tygon tubing lined with Teflon provides for all tube connections.
  • the main chamber is 30 cm long, 21.5 cm wide and 14 cm deep (Figure 8).
  • the chamber has a sealed viewing port assembled on the top side. It also has a port connection to a water reservoir containing 2 1 of sterile water with access to outside air via a stainless steel tube having a PALL 0.2 lm PTFE air filter mounted on it to equalize the pressure with sterile air (Figure 8).
  • the chamber also has an inlet valve allowing for the entry of air that has been passed through an air purifier (charcoal) as well as an air sterilization filter (as described elsewhere herein).
  • At the base of the main chamber is another port with a three-way valve connected to a peristaltic pump via the chamber and an exit port on the right side allowing for water circula- tion through the pump and alternatively as an exhaust to the waste water trap. This easily allows for water to be replaced during the initial leaching processes.
  • a stainless steel column containing 10 g each (in line with each other) of Carbotrap materials A and B (Supelco Co.; Booth et al, 2011, Biotechnol. Lett. 10: 1963-1972, see also U.S. Patent Application No. 13/591,968).
  • the entire system has been built air-tight with the only air access being the air inlet system on the left side of the chamber ( Figure 8).
  • a viewing port allows for monitoring of the events transpiring within the chamber.
  • This microorganism was obtained as an endophyte from the fruit and stems of Citrus aurantifolia (Rutaceae - an ancient plant family genetically related to Sapindaceae) in a subtropical forest of Southeastern Florida in an environment very much resembling that Cretaceous period of Eastern Montana.
  • the fungus was characterized as an Annulohypoxylon sp. on the basis of its ITS sequence identity (99 % level on ca. 831 bp) to other isolates of Annulohypoxylon stygium that had been deposited in Genbank. Its exact species designation is uncertain.
  • the air source was house air that was flowing at 1 1/min through a 4 x 22 cm charcoal air filter in-line with a PALL air sterilization filter and directly into the chamber as described above.
  • Carbo-trapped hydrocarbons were measured gravimetrically using before and after collection weights of the column itself after a 30 min dry purge at 30 °C with a 50 ml/min flow of dry N 2 (Booth et al. 2011). Then, the hydrocarbons were eluted in a programmable oven ranging up to 250 °C over the course of 1 h with dry N 2 at 600 ml/min and with hydrocarbon capturing in a vial bathed in liquid N 2 (Booth et al, 2011, Biotechnol. Lett. 10: 1963-1972, see also U.S. Patent Application No. 13/591,968). The recovered
  • hydrocarbons were weighed and subjected to GC/MS analysis. Hydrocarbons on the "Trap Shale” were measured and obtained in the same manner. This shale carried from 7-11 % (w/w) relative to the amount of "Trap Shale” that was desorbed. The volatiles on both the Carbotrapped and “Trap Shale” samples were determined by SPME-GC/MS analysis. The final data presented were obtained by subtracting all compounds trapped in the control "Trap Shale" from the PBS hosting the fungus.
  • the shale samples from the PBS ("Trap Shale"- treatment and control) were fixed and slowly dehydrated in ethanol, critically dried, coated with gold, and examined with an FEI XL30 SEM Field Emission Gun at 5 kV with high vacuum mode using an Everhart- Thornley detector (Ezra). A gaseous secondary electron detector was used with a spot size of 3, at 15 kV. The temperature was 4 °C with a chamber pressure ranging from 5 to 6 T, providing humidity up to 100 % at the sample. Shale/fossil samples from the field were not dehydrated and subjected directly to gold coating and SEM observations. In this manner, as previously observed, if the fungal hyphae were contemporary, and not appropriately fixed, prior to gold coating, they would collapse under the vacuum in the SEM.
  • Aqueous samples from the PBS that were first diluted 1 :9 v/v (sample to water) and then scanned from 540 to 230 nm in a 1 ml cuvette having a 1 cm light path. The results are reported as total absorbance at 260 nm for each sample.
  • the amount of Carbotrapped hydrocarbons was determined by gravimetric means, after a dry purge, and then desorbed and weighed again (Booth et al, 2011, Biotechnol. Lett. 10: 1963-1972, see also U.S. Patent Application No. 13/591,968).
  • the shale harvested from the "Trap Shale” was also desorbed by heating and trapped in a vial cooled with liquid nitrogen (Booth et al, 2011, Biotechnol. Lett. 10: 1963-1972, see also U.S. Patent Application No. 13/591,968). The results of the experiments are now described.
  • Table 5 A GC/MS analysis of the volatiles being produced on a 10 day old culture of Annulohypoxylon sp. actively growing on a potato/dextrose/agar plate He-tetifess Sitae KeSsii e ai3 ⁇ 4a eiecuJai sseight QssaSiy (caia)
  • Table 6 A GC/MS analysis of the Carbotrapped gases of Annulohypoxylon sp. after a 2 week incubation on a 7 1 culture grown on potato dextrose broth at 22 °C
  • the dry weight of the fungus was 22 g. + Indicates that an authentic control had the same retention and MS as the fungal product
  • the plant materials selected for use in the PBS supported fungal growth very well (Figure 1 1). This included leaves of three plant species selected on the basis of their paleobotanical origins ( Figure 9). Stem pieces of these species were also added to the instrument since paleobotanical evidence has been discovered showing the presence of fossilized fungi in stem tissues in Montana shale near the same location as the shale used in these experiments ( Figure 9).
  • the Annulohypoxylon sp. was capable of colonizing each of the plant materials in the PBS including leaf blades, petioles and stem fragments after a few days of leaching with sterile water.
  • Compounds recovered from the "Trap Shale” in the PBS containing the fungus include representatives of all of the major classes of compounds found in diesel including straight and branched chained hydrocarbons, cyclic alkanes, benzene derivatives and polyaromatic hydrocarbons such as the naphthalenes and azulenes.
  • Reiesiioa iitae Relates sasa Cs -praaiiS MoSecssfe: weigks Qwaiiiy
  • Carbotrap of the control PBS was substantially lower than the fungal counterpart (Table 7). It mostly contained an assortment of aldehydes, ketones, benzenes and furans. These compounds were present in low amounts in the control Carotrap and only a few were the same as those in the fungal PBS-Carbotrap. The source of these volatiles in the Control Carbotrap is apparently related to the autoclaving of the plant materials and changes brought by heating and wetting of the materials.
  • Paleobiosphere This device was constructed to mimic some of the conditions of the ancient earth in order to provide evidence for the hypothesis that the biological degradation (using an endophytic fungus) of plant materials can be an apparent source of hydrocarbons using shale as a trapping medium for the microbial derived compounds. Overall, it appears that the initial experiments to demonstrate this phenomenon have provided some experimental evidence for the hypothesis (Tables 7, 8). Representative compounds in each class of substance in diesel were present in the final "Trap Shale" analysis of the PBS (Table 8). This finding, at least provides a basis for further studies using the PBS in a multitude of different ways.
  • Example 3 An Endophytic Nodulisporium sp. from Central America ProducingVolatile Organic Compounds with Both Biological and Fuel Potential
  • Nodulisporium sp. (Hypoxylon sp.) which has been isolated as an endophyte of Thelypteris angustifolia (Broadleaf Leaf Maiden Fern) in a rainforest region of Central America. It has been identified both on the basis of its morphological
  • the endophyte produces volatile organic compounds (VOCs) that have both fuel (mycodiesel) and use for biological control of plant disease.
  • VOCs volatile organic compounds
  • the most abundant identified VOC was 1,8 cineole, which is commonly detected in this group of organisms.
  • Other prominent VOCs produced by this endophyte include 1-butanol, 2-methyl, and phenylethanol alcohol.
  • antifungal and antibacterial activities were assessed against Staphylococcus aureus, Bacillus subtilis, Candida albicans, Fusarium solani, and Sclerotinia sclerotiorum.
  • small plugs (3 mm diameter) of each test fungi were placed a centimeter away from the edge of a 7-day-old 25-2A culture.
  • the bacterial and yeast cultures were streak-inoculated, starting from the edge of the colony towards the periphery of the plate.
  • the plates were wrapped with Parafilm and incubated at 23 °C or 37 °C for 24-48 h for fungi and bacteria, respectively. Growth of the test pathogens were reported as percent inhibition as compared with their relevant controls.
  • the bacterial and yeast cultures were visually evaluated for the amount of colony inhibition.
  • VOCs produced by 25-2A were obtained commercially.
  • the compounds tested were 2-pentanone; 3-hexanone, 2-4-dimethyl-; 1-butanol, 2-methyl-; 1,8- cineole; propionic acid, 2-methyl-; and farnesene. They were placed in a mixture according to the proportion in which they appeared in the GC/MS profile (see relative areas in Table 10). Aliquots of the mixture ranging from 0.05 to 0.5 ⁇ /ml were placed in pre-sterilized 6 mm micro-cups at the center of the agar surface of a PDA plate having a 50 ml headspace.
  • Test organisms were inoculated on the plates as 3 mm plugs of 7-day-old cultures and on normal PDA plates as controls. Percent inhibition of fungal growth was calculated after 48 h of growth and plotted against the concentration of the mixture per milliliter of the airspace for each organism. The inhibitory concentration yielding 50% inhibition of fungal growth (IC50) for each organism was calculated through extrapolation from the plots (Strobel et al, 2001, Microbiology 147:2943-2950 ).
  • the endophyte ( 25-2A) was grown on gamma- irradiated carnation leaves for 3 weeks to promote the formation of fruiting bodies (Tomsheck et al, 2010, Microbial. Ecol. 60:903-914).
  • the samples were slowly dehydrated in ethanol, critically dried, coated with gold, and examined with an FEI XL30 SEM Field Emission Gun at 5 kV with high vacuum mode using an Everhart-Thornley detector.
  • a gaseous secondary electron detector was used with a spot size of 3, at 15 kV.
  • the temperature was 4 °C with a chamber pressure ranging from 5 to 6 T, providing humidity up to 100% at the sample.
  • ITS 5.8S ribosomal gene 25-2A was grown on PD agar for 7 days and DNA templates were prepared by using the Prepman Ultra Sample Preparation Reagent (Applied Biosystems Inc., USA) according to the manufacturer's guidelines.
  • Universal primer pair ITS 1 5'- TCCGTAGGTGAACCTGCGG-3 '; SEQ ID NO. 1
  • ITS4 5'- TCCTCCGCTTATTGATATGC-3 '; SEQ ID NO. 2 was used to amplify the 18SITS-5.8S region of the fungus by the polymerase chain reaction (PCR).
  • the PCR conditions used were as follows: initial denaturation at 94 °C for 3 min, followed by 30 cycles of 94 °C for 15 s, 50 °C for 30 s, 72 °C for 45 s, and a final extension of 72 °C for 5 min.
  • the 50 ⁇ reaction mixture contained 1 * PCR buffer, 200 ⁇ each dNTP, 1.5 mM MgCl 2 , 10 pmol of each primer, 1-5 ng of DNA, and 2.5 U of Taq DNA polymerase.
  • the amplified product (5 ⁇ ) was visualized on 1% (w/v) agarose gel containing 0.5 ⁇ g/ml of ethidium bromide, to confirm the presence of a single amplified band.
  • the amplified products were purified by Amicon Ultra columns (Millipore, USA) and 10-20 ng were used in a 10 ⁇ sequencing reaction using the Big Dye Terminator sequencing kit (v. 3.1).
  • the forward primer ITS1 (3.2 pmoles) was used in the cycle sequencing reaction, and 20 cycles of 96 °C for 10 s, 50 °C for 5 s, and 60 °C for 4 min were performed in a Biometra Thermocycler.
  • the extension products were purified by ethanol precipitation, dissolved in 15 ⁇ of HiDi Formamide, incubated at 95 °C for 1 min, and loaded on an ABI Prism 377 Genetic Analyzer (Perkin-Elmer, USA) for sequencing.
  • VOCs produced by 25-2A were carried out by the methods described previously (Strobel et al, 2001, Microbiology 147:2943-2950).
  • the organism was grown on PD agar for 10 days in a Petri dish wrapped in a two layers of Parafilm to ensure that the VOCs produced during the entire period of growth accumulated in the headspace of the fungus.
  • a hole was drilled on one side of the plate and a baked SPME fiber (Supelco) 50/30 divinylbenzene/carburen on polydimethylsiloxane on a stable flex fiber was inserted through it and the vapour phase was adsorbed for 45 min.
  • the vapors were injected into a Hewlett Packard 6890 gas chromatograph containing a 30 m x 0.25 mm inner diameter ZB Wax capillary column with a film thickness of 0.50 ⁇ .
  • a thermal program of 30 °C for 2 min followed by an increase to 220 °C at 5 °C/min was applied.
  • Ultrahigh purity helium gas was used as the carrier gas and the initial column head pressure was 50 kPa.
  • the fungus colony is initially creamy white, grows grey from the middle towards the edges as it turns old, and finally becomes brownish, being circular with sharp margins and showing ridges extending from the center to the edge of the colony. Colonies more than one month old turn brownish, with concentric rings forming towards the margin. From the reverse side of the petri plate, the color changes from creamy to light brown to dark brown as the culture grows older, accompanied by heavy sporulation.
  • the colony attains a regular size of 4.5 cm in 20 days.
  • the culture smells strongly with a mix of earthy and fruity odors.
  • the fungus When grown on synthetic media containing individual sugars (sucrose, maltose, sorbitol, lactose, fructose, dextrose, starch, and cellulose), the fungus showed marked variations in cultural characteristics. The organism spreads its hyphae all over the plate in contrast to making a discrete colony with a well-defined margin as it does on PDA. The best growth was observed on maltose (153%) and the lowest on sorbitol (67%) with respect to a PDA control. On water agar and a cellulose-based medium, the growth of the organism was insignificant. The characteristic smell of the volatiles of this organism was diminished when grown on media other than PDA.
  • Nodulisporium sp. which is the anamorphic stage of Hypoxylon sp.
  • 25-2A and Hypoxylon monticulosum strains SUT1 16 formed a sister group with three different species of Daldinia ( Figure 13).
  • the perfect stage of the fungus was not observed on any of the media used for its cultivation.
  • 25-2A is hereafter referred to as Nodulisporium sp.
  • the VOCs displayed selective antimycotic activity against Phytophthora palmivora, Phytophthora cinnamomi, Rhizoctonia solani, and Sclerotinia sclerotiorum rendering them completely inhibited and nonviable except for Phytophthora cinnamomi (Table 9).
  • the other fungal pathogens that were partially inhibited included Pythium ultimum (43%), Aspergillus fumigatus (20%), Botrytis cinerea (20%), and Geotrichum candidum (13%).
  • the VOCs however, showed no inhibition in case of several other bacterial and fungal strains including Candida albicans (Table 1). The latter observation suggests that the co-culture inhibition of C. albicans must be due to a nonvolatile agar diffusible substance(s).
  • the headspace contained several alcohols such as 1-butanol, 2-methyl-; 1-hexanol, 2-ethyl-; phenylethyl alcohol; and 2-naphthalinol, 3-methoxy-.
  • Other VOCs in the mixture included 4- methyl-3-hexanol acetate and propionic acid, 2-methyl-, as well as the important fuel compound cyclohexane, propyl- .
  • Table 10 The composition of VOCs in Nodulisporium sp. as determined by GC/MS.
  • the artificial VOC mixture was most effective against Phytophthora palmivora, Phytophthora cinnamomi, Pythium ultimum, Rhizoctonia solani, Sclerotinia sclerotiorum, and Botrytis cinerea, with an IC50 of 0.1 ⁇ /ml of the headspace for the former three pathogens and 0.15 ⁇ /ml for the latter three.
  • the organisms with the lowest IC50s were also some of the most sensitive to the fungal VOCs (Table 9). All the fungal pathogens tested in this study were susceptible to the artificial VOC mix, with the highest IC50 of 0.35 ⁇ /ml for Geotrichum candidum (Table 9).
  • Nodulisporium sp. was found in nature associated with
  • the fungus was resistant to the VOCs produced by a standard culture of M. albus, which was taken as an indication that it too could be a VOC- producing strain (Strobel et al, 2001, Microbiology 147:2943-2950).
  • This organism was placed as a Nodulisporium/Hypoxylon on the basis of its morphological characteristcs and ITS sequence analysis, although the perfect stage of this fungus was not observed under the conditions used for its cultivation.
  • the VOCs produced by this organism showed differential antimycotic activities, being highly active against Phytophthora palmivora, Phytophthora cinnamomi, Rhizoctonia solani, and Sclerotinia sclerotiorum, and moderately or weakly active against Pythium ultimum, Aspergillus fumigatus, Botrytis cinerea, and Geotrichum candidum (Table 9).
  • the artificial VOC mix significantly mimicked the VOC activity of the endophyte, but the unknown compounds may also have a role in inhibiting other organisms that obviously could not be evaluated (Table 9).
  • the selective activity of the VOCs of this organism may be exploited for inhibition of specific phytopathogens for disease management of agricultural crops. However, it is imperative to learn if this organism can act as a phytopathogen in the crop of interest. Volatile organic compounds are important infochemicals and various fungi produce interesting arrays of compounds that inhibit other microorganisms.
  • VOCs of Nodulisporium sp. is a vast range of compounds that have fuel potential (mycodiesel) (Mends et al, 2012, J. Petrol. Environ. Biotechnol. 3 :3; Riyaz-Ul- Hassan et al., 2012, Microbiology 158:465-473; Strobel et al, 2008; Microbiology 154:3319- 3328; Tomsheck et al, 2010, Microbial. Ecol. 60:903-914). Comparable to the endophyte, also identified as Hypoxylon sp.
  • ⁇ -Farnesene may be also playing a role as an insect repellent, preventing infestation of the host by aphids (Yu et al, 2012, J. Int. Plant. Biol. 54:282-299).
  • the process of natural selection may promote the association of such endophytes with the host plants.
  • the Nodulisporium sp. described herein produces a unique and wide range of bioactive VOCs that also possess fuel potential.
  • the use of molecular biology tools such as epigenetic modulation could be used to further explore the hidden VOC-producing potential of this organism (Riyaz-Ul-Hassan et al, 2008, Microbiology 158:465-473).
  • Such organisms may also be selected as candidates for metabolic engineering and scale-up processes for the production of cost-effective alternate fuels or to find biological utilities.
  • Example 4 An Endophytic Nodulisporium sp. Producing Volatile Organic Compounds Having Bioactivity and Fuel Potential
  • Nodulisporium sp. 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.
  • VOCs volatile organic compounds
  • the organism produces 1, 4-cyclohexadiene, 1- methyl- , 1-4 pentadiene and cyclohexene, l-methyl-4-(l-methylethenyl)- along with some alcohols and terpenoids of interest as potential fuels.
  • Nodulisporium sp. produces a series of alkyl alcohols starting with l-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.
  • a search for new VOC producing endophytes was conducted in the Napo river region of the upper Amazon in Ecuador. At least 20 plants were obtained by clipping terminal stem pieces (ca. 1 x 20 cm) from readily accessible portions of sample trees in a relatively small area of the jungle. The harvested specimens were kept cool and as soon as possible, the stems were surface treated and internal tissue pieces were set out on plates of water agar (WA) and glycerol-arginine medium (GAM) (Tomsheck et al, 2010, Microb. Ecol. 60:903-914). The tissue pieces were incubated at room temperature for several days. Visible fungal growth from the tissue samples were picked as hyphal tips and sub-cultured and transferred to potato dextrose agar (PDA) plates.
  • PDA potato dextrose agar
  • An endophyte of interest was designated EC- 12 and was obtained from a ca. lO m tall leguminous tree-Myroxylon balsamum in the area of S 0.00° 29 ' 960 and W 76° 22 ' 342. This particular organism was not observed as an endophyte associated with any of the other plants that were sampled in this general area.
  • the organism, EC- 12 was stored at -70 °C as No. 2385 in the Mycological Collection of the Department of Plant Sciences at Montana State University. Long term storage of these rainforest endophytes is best done on doubly autoclaved, thoroughly wetted, and leached barley seeds. Scanning electron microscopy
  • SEM Scanning electron microscopy
  • ITS-5.8 S ribosomal gene sequence was carried out by the acquisition of the ITS-5.8 S ribosomal gene sequence.
  • the fungus was grown on PDA for 7 days and DNA templates were prepared by using the Prepman Ultra Sample Preparation Reagent according to the manufacturer's guidelines (Applied Biosystems, USA).
  • the ITS regions of the fungus were amplified with the universal ITS primers ITS1 (5' TCCGTAGGTGAACCTGCGG 3'; SEQ ID NO 1) and ITS4 (5' TCCTCCGCTTATTGATATGC 3'; SEQ ID NO 2) using the polymerase chain reaction (PCR).
  • the PCR conditions used were as follows: initial denaturation at 94°C for 3 min followed by 30 cycles of 94°C for 15 sec, 50C for 30 sec, 72°C for 45 sec, and a final extension at 72°C for 5 min.
  • the 50 ⁇ reaction mixture contained lx PCR buffer, 200 ⁇ each dNTP, 1.5 mM MgC12, 10 pmol of each primer, 1-5 ng of extracted DNA and 2.5 U of Taq DNA polymerase.
  • the amplified product (5 ⁇ ) was visualized on 1% (w/v) agarose gel to confirm the presence of a single amplified band.
  • the amplified products were purified by Amicon Ultra columns (Millipore, USA) and 20 ⁇ 10 ng were used in a 10 ⁇ sequencing reaction using the Big Dye Terminator sequencing kit (v. 3.1), with 2 pmoles of the forward or the reverse primer in the cycle sequencing reaction.
  • the volatiles produced by a 13 day old culture (optimum time) of EC-12 were tested for antimicrobial activity against selected pathogenic fungi according to a VOC bioassay test system previously described for analysis of VOCs produced by Muscodor albus (Strobel et al, 2001, Microbiology 147:2943-2950).
  • the assays were conducted by growing the test organism on one side (half moon) of a Petri plate (on PDA) and then placing a small plug of each test fungus (a 3 mm plug) on the opposite side of the plate (half moon) of a PDA plate. The plate was then wrapped with Parafilm and incubated at 23 °C for varying time periods.
  • test mixture is transferred to a small plastic cup and placed in the center of a Petri plate (PDA) surrounded by small plugs of test organisms and the growth of the test organisms measured after appropriate exposure times. All test organisms were obtained from the Mycological Collection, Department of Plant Sciences, Montana State University.
  • the fungus was grown in 60 ml of PD broth for 7 days in a sealed 250 ml Erylenmeyer flask, with constant agitation at 22 °C. The volatiles were sampled from the headspace of the fungal cultures after days. Briefly, a small hole was drilled in the cap and a preconditioned "Solid Phase Micro Extraction (SPME) fiber" coated with DVB/CAR/PDMS was inserted and the fiber exposed for 5 min.
  • SPME Solid Phase Micro Extraction
  • VOCs bound to the fiber were desorbed for 3 min in a split- /splitless injector (splitless mode, 250 °C) of a Varian 3800 gas chromatograph coupled with an ion trap mass spectrometer attached to a DB5-HT (30 m x .25 mm x 0.25 um). Helium was used as carrier gas at constant flow rate 1 ml/min.
  • the oven program was 30 °C (hold 3 min), 5 C/min to 220 °C, 10 C/min to 250 °C (hold 5 min).
  • the MSD parameters were EI at 70eV, mass range was 30-500 Da, and the scan speed was 2 scans/sec. Headspace SPME- GC/MS was performed on replicate EC- 12 PD broth as well as PD agar cultures.
  • the syringe was then inserted into the splitless injection port of a Hewlett Packard 6890 gas chromatograph containing a 30 m x 0.25 mm I.D. ZB Wax capillary column with a film thickness of 0.50 ⁇ .
  • the column was temperature programmed as follows: 30 °C for 2 min increased to 220 °C at 5°C min "1 .
  • the carrier gas was ultra high purity helium, and the initial column head pressure was 50 kPa.
  • a 30 sec injection time was used to introduce the sample fiber into the GC.
  • the gas chromatograph was interfaced to a Hewlett Packard 5973 mass selective detector (mass spectrometer) operating at unit resolution. The MS was scanned at a rate of 2.5 scans per second over a mass range of 35-360 amu. Data acquisition and data processing were performed on the Hewlett Packard ChemStation software system. In all cases, tentative identification of the fungal compounds was made via library comparison using the NIST 2011 (US- National Institute of Standards and Technology) mass spectral library and all chemical compounds described in this report use the NIST data base chemical terminology. Only compounds, having a quality match score better than 70%, are listed and described in this report. Peak areas for all other unidentified compounds were lumped and summed for each EC- 12 culture tested. Compounds appearing in the control flasks, plates, or tubes were removed from the analysis.
  • the endophytic organism EC- 12 by light and scanning electron microscopy produced an imperfect stage resembling that of Nodulisporium sp. ( Figure 14).
  • the organism when grown on PDA (2 week old culture) produced whitish felt-like mycelia on the 4 cm perphery of the culture.
  • the center of the culture was a distinctively greyish/brown coloration.
  • the mycelium produced erect to suberect, branching conidiophores with slender conidiogenous cells attached in irregular to verticillate patterns. Conidiogenous cells terminated in clusters of one-celled conidia at the apical end.
  • Table 1 1 The VOC composition of Nodulisporium sp. flask analyzed at 7 days of growth by Headspace SPME-GC/MS on three different cultures each grown on PD broth.
  • Table 12 GC/MS for liquid collected from the Carbotrap extraction of Nodulisporium sp. produced by 21.5 g dry weight of fungal hyphae over a 2 week period.
  • VOC stream via such compounds as oximes and thiazoles
  • nitrogen and sulfur are undesirable for fuel use. Therefore, overall, it appears that almost the entire mixture of VOCs produced by this fungus has fuel potential.
  • the scope of the VOC stream may potentially be tailored for particular combustion applications.
  • Table 13 Effects of a 13 -day-old colony of Nodulisporium sp. on various fungi in a simple split plate test
  • VOCs of Nodulisporium sp. could be recovered by the Carbotrap technology, they too were subjected to a bioassay test utilizing several select plant pathogens (Table 14).
  • Table 14 Effects of liquid volatile compounds collected from a 13-day-old colony of Nodulisporium sp. on various fungi as conducted in a well assay on a Petri plate with PDA
  • the liquid mixture yielded the greatest inhibition response from R.solani and S. sclerotiorum but with somewhat mixed results from the other test organisms (Table 14). This appears to be the first report where by fungal VOCs have been trapped and reconstituted in bioassay tests.
  • the decrease in the relative bioactivity may be related to the small amount of materials tested, a loss of one or more critical bioactivity compounds, or the lability of certain components that are necessary for bioactivity.
  • the fungal isolate used in this study was obtained as an endophyte from the upper Amazon basin.
  • the organism by virtue of its molecular sequence data and its morphology best fits the description of a Nodulisporium sp. with a Daldinia sp. as its most likely perfect stage ( Figures 14 and 15).
  • This isolate of Nodulisporium sp. produces a series of VOCs many of which are hydrocarbons or hydrocarbon derivatives (Tables 11 and 12).
  • the composition of VOCs seems to be related to the conditions under which the fungus is grown (Tables 1 1 and 12). For instance, the cyclohexene derivatives are consistently produced when the organism has been grown on a solid medium or under microaerophilic conditions (Table 1 1).
  • Example 5 Ti-13, a Nodulisporium sp. isolate
  • Ti-13 a Nodulisporium sp. isolate, was isolated from Cassia sp. in the hills of western Thailand. Under SEM, Ti-13 looks identical to a Nodulisporium sp. ( Figure 16). The spores of Ti-13 are 5-6 microns x 2.5 ⁇ 4 microns ( Figure 17). Dendogram analysis confirms that Ti- 13 is a Nodulisporium sp. ( Figure 18). Ti-13 was found to produce 1,8-cineole, in addition to other VOCs (Table 15). Methods of isolating and analyzing VOCs produced by fungi are described elsewhere herein.
  • Table 15 VOCs produced by Ti-13, a Nodulisporium sp. isolate
  • Example 6 Th-9. a Nodulisporium sp. isolate
  • Th-9 a Nodulisporium sp. isolate, was isolated from pomelo in the hills of western Thailand. Under SEM, Th-9 looks identical to a Nodulisporium sp. Th-9 was found to produce 1,8-cineole, in addition to other VOCs (Table 16). Methods of isolating and analyzing VOCs produced by fungi are described elsewhere herein.
  • Table 16 VOCs produced by Th-9, a Nodulisporium sp. isolate Retention Time ⁇ min) Relative Area Possi le ompsnind Quality Mas (Pal relative area
  • Example 7 Fl-9. a Nodulisporium sp. isolate
  • Fl-9 a Nodulisporium sp. isolate, was isolated from Taxodium distichum from a swamp in Florida. Under SEM, Fl-9 looks identical to a Nodulisporium sp. Fl-9 was found to produce 1,8-cineole, in addition to other VOCs (Table 17). Methods of isolating and analyzing VOCs produced by fungi are described elsewhere herein.
  • Table 17 VOCs produced by Fl-9, a Nodulisporium sp. Isolate

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Abstract

L'invention porte sur un champignon isolé ayant le stade imparfait de Nodulisporium. Le champignon isolé produit au moins un composé choisi dans le groupe constitué par le 1,8-cinéole et le 1-méthyl-1,4-cyclohexadiène. L'invention porte également sur un procédé pour la production d'au moins un composé choisi dans le groupe constitué par le 1,8-cinéole et le 1-méthyl-1,4-cyclohexadiène. Le procédé comprend la culture d'un microorganisme sur un milieu de culture ou au sein de celui-ci dans un récipient dans des conditions suffisantes pour produire ledit ou lesdits composés.
PCT/US2014/024650 2013-03-12 2014-03-12 Microorganismes pour la production de composés organiques volatils et procédés les utilisant WO2014165174A2 (fr)

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JO3416B1 (ar) * 2009-04-27 2019-10-20 Jeneil Biosurfactant Co Llc تركيبات مضادة للبكتيريا وطرق استخدامها
US9090921B2 (en) * 2010-05-18 2015-07-28 Gary A. Strobel Method of producing volatile organic compounds from microorganisms
EP2571995A4 (fr) * 2010-05-18 2014-01-22 Gary A Strobel Système et procédé d'obtention de composés organiques volatils à partir de champignons
US9222096B2 (en) * 2011-05-23 2015-12-29 Agriculture Victoria Services Pty Ltd Fungi and products thereof

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CN105861334A (zh) * 2016-06-22 2016-08-17 福建农林大学 一株能促进台湾相思生物量增长的内生真菌
CN105861334B (zh) * 2016-06-22 2019-06-04 福建农林大学 一株能促进台湾相思生物量增长的内生真菌
CN106754399A (zh) * 2016-11-18 2017-05-31 广西壮族自治区药用植物园 暗色环纹炭团菌的分离方法
CN106754399B (zh) * 2016-11-18 2020-03-24 广西壮族自治区药用植物园 暗色环纹炭团菌的分离方法

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