Chemical Analysis of Hydrocarbon Secretions from the Fungus Ascocoryne sarcoides
By: Justin Earley, Advisor – Dr. Holly Ziobro
University of Wisconsin – Platteville , Department of Chemistry
Co-authors:
Michelle Hendricks, University of Wisconsin – Platteville
Dr. Elizabeth Frieders, University of Wisconsin – Platteville, Department of Biology
Dr. Gary Strobel, Montana State University, Department of Biology
Abstract
The protein abundance in the fungus Ascocoryne sarcoides was attempted to be shifted to create
more favorable hydrocarbon secretions such as octane, heptane, and 2-pentene using variant metal
ion concentrations introduced to the growth medium. Colonies of Ascocoryne sarcoides were
grown in closed specimen tubes containing oatmeal agar with variant concentrations of the ions;
iron (3+), coper (2+), and zinc (2+) and an activated carbon strip. After a growth period of three
weeks, the activated carbon strips were prepared in clamped vials with carbon disulfide as a
solvent to be analyzed using gas chromatography–mass spectrometry.
Introduction
Fuel has been a concern for many years all over the world resulting in a push to find a renewable
fuel source. In the past, research has been performed on corn and algae to aid in lowering the
consumption of petroleum. However, major problems have surfaced with both corn and algalbased fuel. Corn requires many acres of land and the percent of oil returned per cubic meter is
very low. Algae produces an approximate maximum of 77% oil per cubic meter yield, but
demands high amounts of nitrogen (Demirbas et. al., 2011). In fact, the amount of nitrogen
needed to grow enough algae to replace 5 % of the petroleum usage in the United States is twice
the amount used to grow all food crops in the United States currently. (Biofuels: What are They?)
Furthermore, both algae and corn must undergo chemical processing to turn plant lipids, the
molecular structures that make up fats, into burnable fuel. The chemicals needed for this
processing are volatile and linked to peripheral nervous system failure after prolonged exposure.
A discovery, by Dr. Gary Stobel of Montana State University, of the fungus Gliocladium roseum,
now classified as Ascocoryne sarcoides (A. sarcoides), is a promising solution to the petroleum
problem. A. sarcoides can turn cellulose, the most abundant sugar molecule in organisms,
directly into hydrocarbons; the molecular structure group that gasoline and diesel consist of
(Strobel et. al., 2008). By converting cellulose directly into hydrocarbons the chemical processing
step needed to produce fuel from corn or algae is eliminated, thus cutting costs and safety
hazards.
Research on A. sarcoides has shown that it can feed off plant waste to produce hydrocarbons
(Strobel et. al., 2008). A. sarcoides could be used to break down plant waste from farms and
industry in order produce hydrocarbon biofuel. Currently this is a step in the right direction
towards solving the world’s energy crisis. However, little is known about A. sarcoides and in
order to utilize the fungus, more exploration must be carried out on how this fungus functions in
different environments.
In 2003 a study was published on the effects of metal ions on the RNA transcription of fungal
growths. The study discovered that when trace metals where introduced into the soil around a new
fungal growth; the RNA would work more efficiently or less efficiently based on the absence or
presence of some metal ions. The most effective metal ions they discovered was copper 2+
(Cu2+), iron 2+ (Fe2+), and zinc 2+ (Zn2+). If the RNA transcription levels are altered, that may
lead to changes in the protein profile of A. sarcoides. In turn, a change in the abundance
of volatile compounds produced by A. sarcoides may be observed.
Experimental
Methods and Material
All cultures prepared in the experiment were made with a base of oatmeal agar which was
prepared from commercially available material. Gum agar (Fisher Scientific, USA) was
combined with and oatmeal enhanced water made by boil 24 grams of whole grain old fashion
oats (Quaker, USA) in 600 mL of de-ionized water for two minutes. Following boiling the
oatmeal and water solution was then separated using a C5 Centrifuge: C5C-08SU-15T3 (LW
Scientific, USA) ran at 700 rpm for three minutes. The liquid layer was then poured off and used
to create the growth medium. Ion solutions were made from the salts of Fe2(SO4)3, MnCl2,ZnSO4,
and CuCl2 and pipetted as described below and in figure 1.1 to study how the trace metal ion’s
concentrations effect the amounts of volatile compounds produced by A. sarcoides.
Trace metal ion sample’s concentration mixtures:
Cu2+ Variation: CuCl2 was dissolved in deionized water as the source of Cu2+ for all
solutions. Five Cu2+ concentration mixtures were made from 6.32∙10-8 g/mol to 6.32∙10-4
g/mol which increased in intervals of a power of ten. Only the concentration of Cu2+ was
altered for these samples of the study. Fe3+ was added to all Cu2+ variations at a
concentration of 7.19∙10-6 g/mol, Zn2+ was added to all Cu2+ variations at a concentration of
6.98∙10-6 g/mol, and Mn2+ was added to all Cu2+ variations at a concentration of 5.29∙10-6
g/mol.
Fe3+ Variation: Fe2(SO4)3 was dissolved in deionized water as the source of Fe3+ for all
solutions. Five Fe3+ concentration mixtures were made stating at 7.19∙10-8 g/mol to 7.19∙10-4
g/mol which increased in intervals of a power of ten. Only the concentration of Fe2+ was
altered for these samples of the study. Zn2+ was added to all Fe2+ variations at a
concentration of 6.98∙10-6 g/mol and Mn2+ was added to all Fe2+ variations at a concentration
of 5.29∙10-6 g/mol.
Zn2+ Variation: ZnSO4 was dissolved in deionized water as the source of Zn2+ for all
solutions. Five Zn2+ concentration mixtures were made stating at 6.98∙10-8 g/mol to 6.98∙10-4
g/mol which increased in intervals of a power of ten. Only the concentration of Zn2+ was
altered for these samples of the study. Fe3+ was added to all Zn2+ variations at a
concentration of 7.19∙10-6 g/mol and Mn2+ was added to all Zn2+ variations at a
concentration of 5.29∙10-6 g/mol.
A standard concentration mixture was created using Fe3+ at a concentration of 7.19∙10-6
g/mol, Zn2+ at a concentration of 6.98∙10-6 g/mol, and Mn2+ at a concentration of 5.29∙10-6
g/mol. All concentration mixtures were added to the oatmeal agar medium. Each
concentration variation was produced in triplicate using specimen tubes that are sealed using
screw caps and all specimen tubes were autoclaved as to sterilize the testing environment
once filled with their corresponding ion mixtures.
Table 1.1 Sample testing combinations. Fourteen sample combinations in total with each being tested in
triplicate.
Concentrations Fe(3+)
10(-8)
10(-7)
Fe(3+) Variations
Zn(2+)
Cu(2+)
Mn(2+)
1
10
Zn(2+) Variations
Zn(2+)
Cu(2+)
1,2,3,4
1,2,3,4
6
10(-6) 5,6,7,8
5,6,7,8
10(-5)
3
10(-5)
7
10(-4)
4
10(-4)
8
Concentrations Fe(3+)
10(-8)
Cu(2+) Variations
Zn(2+)
Cu(2+)
Mn(2+)
9
Standard
Zn(2+)
Concentrations Fe(3+)
10(-8)
10(-7)
10
10(-7)
10(-6) 9,10,11,12,13 9,10,11,12,13
11 9,10,11,12,13
10(-6)
10(-5)
12
10(-5)
10(-4)
13
10(-4)
Mn(2+)
5
10(-7)
2
(-6)
Concentrations Fe(3+)
10(-8)
14
Cu(2+)
Mn(2+)
14
Culture of A. sarcoides was acquired from the Montana State University and was preserved on
barley seeds for storage and delivery. The barley seeds were cultured in petri dishes containing
potato dextrose agar (Fisher Scientific, USA) as suggested in “Genomic Analysis of the
Hydrocarbon-Producing, Cellulolytic, Endophytic Fungus Ascocoryne sarcoides” (Gianoulis TA
et. al., 2008) at room temperature and humidity. Proper sterilization techniques were used to
ensure no contamination in amongst the A. sarcoides colonies. The colonies grown in petri dishes
were the source for all further culturing in the specimen tubes.
Proceeding autoclaving of the specimen tubes, the contents were allowed to cool to room
temperature before handling. A culture of A. sarcoides approximately one centimeter by one
centimeter was introduced into the specimen tubes which contained oatmeal agar and the variant
concentrations of ions based on the mixtures previously stated. Once A. sarcoides was introduced
into a specimen tube, a one centimeter by three centimeter activated carbon strip was attached in
the headspace of the specimen tube. The caps of the specimen tubes were tightly screwed on and
parafilmed to prevent leaking of any volatile compounds produced by A. sarcoides. All the steps
were performed in a clean hood that was cleaned with 70% ethanol as to prevent contamination
to the A. sarcoides colonies. The colonized specimen tubes were incubated at room temperature
and humidity for a period of three weeks.
After incubation, the specimen tubes were placed in an 80 degree Celsius oven for three hours to
aid in the vaporization of any gases produced by A. sarcoides. The activated carbon strips were
then removed from the specimen tubes and placed in a GC-MS vial along with 50 milliliters of
carbon disulfide; CS2. The vials were then crimped closed and stored until GC-MS analysis could
be ran.
14
Figure 1.1 Cultured specimen tube set-up.
Testing
Gas chromatography–mass spectrometry (GC-MS) would be used to give a comparative analysis
of the volatile gas compounds produced by A. sarcoides and captured in the activated carbon
strips during incubation. The three compounds octane, heptane, and 2-pentene, proven to be
produced by A. sarcoides when grown using oatmeal agar (Strobel GA et. al., 2008), will be the
principal compounds of interest due to their wide use and low boiling points as related to the other
volatile compounds produced by A. sarcoides. Octane would be introduced to the vials containing
the activated carbon strip and CS2 to provide a standard reference concentration to calculate the
concentration of octane, heptane, and 2-pentene in the activated carbon strips.
In the case that a correlation is found between the trace metal ion concentration and the amount
of volatile compounds produced, one and two dimensional electrophoresis will be used to
qualitatively observe the change in protein abundance and confirm the assumption that the trace
metal ions alter the amount of proteins made by A. sarcoides.
Results
The results are inconclusive until GC-MS analysis can be ran.
A variant in the amount of octane, heptane, and 2-pentene is predicted to be observed. The
increase or decrease of octane, heptane, or 2-pentene would show a correlation with the
concentration of the trace metals in the growth medium. From this data a mixture containing the
most favorable concentrations would be made and tested to see if the production of octane,
heptane, and 2-pentene could be optimize.
One dimensional electrophoresis was preformed to see if it was possible to see the protein bands in
A. sarcoides.
Figure 2.1 One dimensional electrophoresis gel with proteins extracted from A. sarcoides. The protein bands can be
seen as faint lines near the top of the gel.
Protein bands were observed in the one dimensional electrophoresis gel; however, these bands
were not as prevalent as expected and fainter in appearance as expected.
Discussion
A. sarcoides presented many challenges to grow and maintain the culture. These were the initial
hurtles that had to be overcame before the start of and sample testing. A. sarcoides has a pinkish
grey color to it once it begins to grow its sexual reproduction spores. In order to test that a sterile
culture of A. sarcoides was being maintained a culture analysis of A. sarcoides grown in a petri
dish on PDA medium was performed by Professor Elizabeth Frieders PhD, University of
Wisconsin-Platteville depart of biology. During her investigation of the colonized A. sarcoides
petri dishes, Dr. Frieders noted little to no contamination amongst or around the A. sarcoides
colonies. Dr. Frieders then cross checked this with the literature to confirm that the spores that
were being produced by the A. sarcoides culture were in fact that of A. sarcoides. The cross
reference came back positive thus confirming that the sterilization techniques being used were
sufficient.
A. sarcoides was then cultured in a petri dish on standard oatmeal agar to compare the growth
cycle to that of PDA. After an incubation period of three weeks it was noted that the A. sarcoides
colonies growing on the oatmeal agar medium were much more translucent and dull in color as
compared to the colonies grown on PDA. However, growth was still seen but at a much slower
rate and the development of asexual spurs happened four weeks after the PDA colonies occurred.
The test samples were still grown using oatmeal agar medium as this was stated from previous
studies to provide the best volatile compound yield (Strobel GA et. al., 2008). A conclusion about
the volatile compound production of the oatmeal agar medium colonies in comparison to PDA
colonies could not be made without GC-MS testing and so the tests were continued based on the
published article Gary Strobel PhD.
Figure 3.1 Reproduction cycle of A. sarcoides. The time in which the cycle takes depends on growth conditions.
The original instructions for mixing oatmeal agar used Fe2+ as a trace metal ion. Fe3+ was used
instead due to the speed in which Fe2+ oxidizes. This quick oxidation makes it very difficult to
maintain Fe2+ in and aqueous state. The effect of using Fe3+ is unknown because no GC-MS
analysis was completed.
When A. sarcoides was cultured in the specimen tubes the sample growth pattern as the culture
grown in the petri dish on oatmeal agar was noted. This was predicted; however, this similarity
confirmed that the changing of the trace metal ion’s concentration did not affect the reproduction
cycle of A. sarcoides.
Conclusion
Do to UW-Platteville’s GC-MS malfunctioning, a final conclusion cannot be drawn. The testing
of the collected volatile compounds from A. sarcoides will be performed at a later date when the
instrument is functioning again.
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