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The isolation of a red pigment, having industrial and medicinal potential
Abstract
In 2006, the first instance of White-Nose Syndrome was documented in upstate New York (1,2).
Investigators found that bats in the North Eastern United States were dying at a tremendous rate. The
cause of their deaths was initially unknown but later identified as the fungus Pseudogymnoascus
destructans (1,2). This psychrophilic Ascomycete colonizes the muzzle and other exposed skin areas of
several species of hibernating bats and can cause up to 100% mortality (3) in hibernacula. The initial
spread of this pathogenic fungus is believed to be transmitted by way of both professional and
recreational cavers, visiting multiple caves with inadequately cleaned gear (1). The major aims of
research pertaining to P. destructans includes efforts to control the spread and mortality associated with
WNS, and identify and isolate metabolites from P. destructans with potential medical and industrial
relevance. Namely, a rubicund pigment found diffusing throughout the agar under various
environmental conditions. The aim of our research is to isolate and identify the pigment that is
produced by P. destructans. The tools necessary to meet this challenge include protocols from both
biology and chemistry. While we characterize the growth requirements and secondary metabolite
production of this organism using biological understandings, we also employ chemical techniques during
the various processes leading to isolation. An isolated pigment may play a significant role in the
development and progression of WNS in bats and potentially have broad industrial uses as red pigment
is rarely found in nature. The isolation of an industrially employable pigment holds great value.
Introduction
The focus of this study was to isolate and identify a red pigment, found diffusing throughout the
agar where P. destructans is present. Using chemical isolation techniques such as TLC, HPLC, and Mass
Spectroscopy we are able to gain a better understanding of the components associated with the
diffusible pigment. P. destructans culture is also an integral part of this study, but not the primary focus
of this particular entry, as pigment isolation and characterization is the aim discussed herein.
Experimental Details
Growth of P. destructans, Origin Of Pigment Samples
P. destructans spores were grown on Sabouraud Dextrose Agar and L-DOPA enriched media at
15° C for a minimum of two weeks before significant fungal growth and pigment production was
identified.
Pigment Extraction from Mycelia
Using a sterile laboratory utensil, the fungal mycelia were scraped off of the agar from the
aforementioned plates. The mycelial scrapings were washed with ethanol and placed in 50-ml Falcon
tubes. To these tubes, NaOH was added. The mycelia in NaOH were stored in a refrigerated space for
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the extraction to take place. Following this extraction, the supernatant was filtered off and additional
NaOH was added to the mycelia in the 50-ml Falcon tubes. Again, the mycelia in NaOH were stored in a
refrigerator for additional extraction. From this second extraction, the supernatant was filtered off and
combined with the supernatant from the first extraction.
Separation of pigment from NaOH supernatant
An aliquot consisting of 1 part supernatant and 2 parts HCL, was combined at room
temperature. This reaction of NaOH and HCL resulted in NaCL and H2O in addition to the pigment and
any unknown compounds present. The product resulting from the HCL treatment was initially left in the
fume hood until water and pigment remained. We later tried two methods in order to distinguish the
best technique to remove the water. The first method employed was evaporation with applied heat.
This was done by placing our sample in a beaker set on a hot plate in order to boil off the water. The
second method employed was lyophilization. Our sample was stored in a 50-ml Falcon tube and placed
in a -80° C freezer. The frozen sample was placed in the lyophilizer until dry.
HPLC Analysis
The following HPLC tests were run on a C18 non-polar, reverse phase column. The solvents used were
acetonitrile and water. In all samples, acetonitrile was ramped to various concentrations, with water
as the remaining solvent percentage at any point in time. The column was set to run at 1ml of solvent
system per minute. All samples injected were 10ul in volume and analyzed for all wavelengths
between 200 – 350nm.
First HPLC Time Program
We injected each of the following samples into the HPLC machine. The total run time was 60 minutes.
Within the initial 5 minutes of the run, acetonitrile ramped from 0% to 2%. From the 5 th minute to the
45th minute, acetonitrile was ramped from 2% to 98%. From the 45th minute to the 50th minute, the
concentration of acetonitrile remained stationary at 98%. From the 50th minute to the 55th minute, the
concentration of acetonitrile ramped down from 98% to 2%. The acetonitrile concentration remained
constant at 2% for the remaining 5 minutes until the end of the run.
Sample name: MX
Origin: Mycelial pigment originally stored in NaOH.
Sample name: Red Pigment
Origin: Pipetted red droplets, having developed on top of the colony
Sample name: Gold Pigment
Origin: Pipetted gold droplets, having developed on top of the colony
Sample name: Brown Pigment
Origin: Pipetted brown droplets, having developed on top of the colony
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Second HPLC Time Program
We injected 10 ul of each of the following samples into an HPLC machine. Over the span of 120 minutes
the column ran at 1 ml per minute. Acetonitrile was ramped from 0% to 2% in the first 5 minutes. At the
60 minute mark, acetonitrile was at 98% and remained stationary for 5 minutes. At the 65 th minute of
the run, acetonitrile ramped down to reach 2% at the 115 minute mark. From the 115 minute mark to
120 minutes, acetonitrile remained at 2%, at which point the run ended.
Sample name: Red Pigment
Origin: Pipetted red droplets, having developed on top of the colony
Sample name: Synthetic Melanin
Origin: Synthetic Melanin from commercial provider
TLC Analysis
Our TLC analysis was done on three sample groups. Group 1 was the red pigment derived from the
washed mycelia, initially extracted by NaOH. Group 2 is from the pigmented droplets collected directly
from the organism.
Group 1
Our negative control was NaOH, with a positive control being synthetic melanin diluted to 10 -2. The
extracts derived from P. destructans were labeled LT01A, LT02A, and DK01A for Light, Light, and Dark
growing conditions respectively. All samples were spotted on polar TLC plates with MeOH, Acetone,
Benzene, and Hexane.
Group 2
The red, gold, and brown droplets were collected directly from P. destructans and spotted on a polar
TLC plate with Acetone as the solvent.
Group 3
Sample R+G was a mixture of red and gold droplets while sample G was exclusively golden droplets. The
positive control was synthetic melanin diluted to 10-2. These three samples were spotted on a polar TLC
plate with a solvent system of 5 Ethyl Acetate: 2 Acetone: 1 MeOH.
Mass Spectroscopy
The red, gold, and brown droplets, having been directly collected from the organism P. destructans were
filtered and submitted for mass spectroscopy analysis. In addition, synthetic melanin was submitted
alongside the aforementioned samples to serve as a positive control in the instance that characteristic
peaks for melanin were present in the red, gold, and brown droplet samples. The mass spectral analysis
conducted was an ESI(+) pre screening with direct infusion. LC-MS was also run, using the 120 minute
time program developed for the HPLC.
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Disk Diffusion Assay
Disks were prepared by depositing a 20ul aliquot per disk of the following.
Sample name: R+G
Origin: Red and gold droplets combined, collected from the top of the colony.
Sample name: G
Origin: Gold droplets, collected from the top of the colony.
Sample name: Synthetic Melanin
Origin: Commercially acquired synthetic melanin diluted to 10-2.
Sample name: Tobramycin
Origin: Commercially acquired positive control.
Sample name: Acetone
Origin: Laboratory solvent used as negative control.
The aforementioned disks were placed on Muller Hinton agar and run in triplicate with a lawn of
Escherichia coli, Staph aureus, and Acinetobacter baumannii respectively. Before inoculation, all
organisms were diluted to the McFarland standard. All plates were inoculated at 37° C for 48 hours.
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Results
Growth of P. destructans, Origin of Pigment Samples
As seen in the images below, pigment production was detected in both SDA and L-DOPA enriched
media.
Image 1
P. destructans on SDA Image 2
P.destructans in L-DOPA enriched SDA
Separation of pigment from NaOH supernatant
The NaOH that was treated with HCL, resulted in a mixture of salt, water, pigment, and unknowns.
Image 3 displays the heated sample that maintained its color, but appeared burnt. However, the
lyophilized sample, seen in Image 4, resulted in bands of salt and pigment that were preferable to use
for analysis.
Image 3
Heated sample Image 4
Lyophilized sample
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HPLC Analysis
The Chromatogram, labeled #A, shows the amplitude of spectral absorption over time. The Spectral
Max Plot, labeled #B shows the amplitude of absorption over time for all wavelengths between 200 –
350nm. The time program is included as a reference for acetonitrile / water ratios at any given point
in time.
Sample name: MX
The aforementioned lyophilized sample was named MX and resulted in the following graphs.
HPLC Graph 1A
Chromatogram
60 Minute Time Program
HPLC Graph 1B
Spectrum Max Plot
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Sample name: Red
HPLC Graph 2A
Chromatogram
60 Minute Time Program
HPLC Graph 2B
Spectrum Max Plot
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Sample name: Gold
HPLC Graph 3A
Chromatogram
60 Minute Time Program
HPLC Graph 3B
Spectrum Max Plot
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Sample name: Brown
HPLC Graph 4A
Chromatogram
60 Minute Time Program
HPLC Graph 4B
Spectrum Max Plot
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Sample name: Red
HPLC Graph 5A
Chromatogram
120 Minute Time Program
HPLC Graph 5B
Spectrum Max Plot
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Sample name: Synthetic Melanin
HPLC Graph 6A
Chromatogram
120 Minute Time Program
HPLC Graph 6B
Spectrum Max Plot
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TLC Analysis
For TLC 1, samples LT01A, LT02A, and DK01A are seen below, synthetic melanin as the positive control
and NaOH as the negative control. MeOH resulted in the best separation for all extractions.
TLC 2, consisted of various gold droplets and synthetic melanin as a control on far left.
TLC 3 was spotted with red+gold, gold and synthetic melanin on the far right. The solvent system used
was 5 Ethyl Acetate : 2 Acetone : 1 Methanol.
TLC 1
TLC 2
TLC 3
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Mass Spectroscopy
The following mass spec results represent all masses in the respective samples, without HPLC
separation.
Mass Spec 1
Solvent (50%MeOH+0.1%HCOOH)
Mass Spec 2
Synthetic Melanin
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Mass Spec 3
Brown
Mass Spec 4
Gold
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Mass Spec 5
Red
Disk Diffusion Assay
Of organisms tested in the disk diffusion assay, only the positive control, Tobramycin, resulted in zones
of bacterial inhibition for E. coli, S. aureus, and A. baumannii.
Discussion
In seeking to isolate and identify the red pigment, we began by growing the P. destructans
organism on various media, including L-DOPA enriched media. However, as we see in images 1 and 2,
the SDA plates and L-DOPA enriched SDA plates both exhibit pigment production. All three pigment
colors have been identified on SDA, PDA and L-DOPA enriched plates. Our reasoning behind using LDOPA, was to provide our organism with metabolic building blocks for melanin production. While
quantifying pigment production has been a challenge, there does not appear to be any significant
increase in pigment production in our organism when growing with L-DOPA.
Of the red pigment extracted in NaOH, it appears that the treatment with HCL has successfully
resulted in water and salt. In removing the water associated with this reaction, lyophilization maintains
the integrity of the red pigment as opposed to evaporating with heat, which resulted in a darker
pigment, burnt in appearance. However, lyophilization is limited to small quantities as opposed to
evaporation with heat.
MeOH provided the best TLC separation for the red pigment initially extracted in NaOH.
However, synthetic melanin did not run regardless of the solvent or solvent system used. Of the
pigmented beads taken directly from the fungus, only one TLC was possible for all pigments due to an
initial decrease in production of red and brown-pigmented beads. As new organisms were plated, the
volume of available pigment increased. However, the production of the red and brown pigments was
not as prevalent as the production of gold pigment. In pipetting pigment droplets from our organism, we
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encountered particular difficulty in differentiating between red and gold pigments due to a gradient in
shades. To be conservative in labeling, we named our red sample as R+G due to the possibility of gold
pigment being present in the lighter shades of red. However, due to the high volume of golden pigment
available, we were able to collect a sample known as G, consisting exclusively of that hue. Using samples
R+G and G, we were able to develop an improved solvent system, revealing 4 fractions visible in ultra
violet light.
HPLC analysis of the lyophilized sample, also known as MX, resulted in a graph with five
particularly strong peaks. As a comparison, we ran dilutions of the red, gold, and brown pigment
exudences found as beads on the top of the P. destructans colony. In comparing the HPLC output for
both MX and the various colored beads, we sought to identify common spectral absorbances between
the two. However, the red, gold, and brown bead absorptions in our 60 minute runs differ significantly
from the MX sample derived from the extracted mycelia. A possible reason for this difference is the
dilute nature of the collected beads, verses the concentrated pigment resulting from lyophilization. Also,
residual salts may have contributed to the differences in the HPLC output.
For the red, gold, and brown bead, a large peak was found eluting off of the column towards the
end of each run. In order for the chemicals associated with that peak to elute off of the column with
good separation, the run time was lengthened to 120 minutes with acetonitrile slowly ramping up and
down in concentration during that time. The sample, identified as red, when run on a 120 minute
program, resulted in an absorption pattern similar in appearance to that of the synthetic melanin run at
120 minutes.
Seeing this similarity in HPLC outputs, we submitted 4 samples for mass spectroscopy. While
HPLC readings for L-DOPA melanin appear similar to the red pigment, mass spectroscopy shows
different masses for the two samples, suggesting that the red pigment is not L-DOPA melanin. However,
this does not suggest that the red pigment is in no way a melanin. Since melanins have various
structures, it is possible that the red pigment tested is a different kind of melanin than L-DOPA.
However, similarities between the red, gold, and brown pigments suggest shared compounds between
the three exuded colors. Mass spectroscopy database searches for each sample, resulted in large
quantities of possible compounds. HPLC separation before mass spec analysis is likely to result in more
succinct database outputs.
In order to determine potential antimicrobial activity, disk diffusion assays were used with
samples R+G, G, and synthetic melanin. Of the organisms tested, none appeared to be inhibited by the
fungal extracts or synthetic melanin. Disk diffusion with different organisms may reveal antimicrobial
activity. However, due to the limited quantities of pigment available for experimentation, disk diffusion
assays are not always feasible.
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Conclusion
When grown in both dark and illuminated conditions, P. destructans appeared to produce the
same amount of pigment. L-DOPA enriched SDA plates did not produce any more pigmentation than the
SDA plates. Furthermore, Mass Spectroscopy reveals similar masses shared between the red, gold, and
brown pigments. While HPLC graphs suggest strong similarities between synthetic melanin and our red
pigment, Mass Spectroscopy suggests otherwise. However, this does not eliminate melanin as possibly
being the pigment; it does however suggest that the red pigment is not L-DOPA melanin. In addition to
isolating the red pigment, our intent was also to identify potential medicinal and industrial uses for the
various extracts of P. destructans. Initial testing has revealed that that E. coli, S. aureus, and A.
baumannii are not inhibited by the extracts used in disk diffusion. However, testing against additional
organisms may reveal antimicrobial activity.
Future work
With more pigment production, additional TLC tests could be run on all 3 pigments, providing
more samples to use in the development of solvent systems. Also, LC-MS analysis would be beneficial,
particularly when comparing results to known pigments (4). Concentrated samples with improved HPLC
protocols, should produce graphs with stronger peaks, necessary for LC-MS. Once a mass of interest has
been identified, accurate mass can be run on the samples, providing further clarity on the what
compounds are present.
In reference to medicinal potential and pigment production, additional disk diffusion assays
could be performed, potentially inhibiting pathogenic organisms. P. destructans could be exposed to
varying amounts of UV light, causing a stress that may induce greater pigment production. Growing P.
destructans alongside another organism may change the behavior of the colony. In addition, volatiles
from organisms grown nearby may also play a role in inducing particular metabolic activity in the P.
destructans colonies.
References
1. Blehert D, Hicks A, Stone W, et al. Bat white-nose syndrome: an emerging fungal pathogen?. Science
(New York, N.Y.) [serial online]. January 9, 2009;323(5911):227. Available from: MEDLINE with Full Text,
Ipswich, MA. Accessed October 25, 2014
2. Chaturvedi V, Springer D, Chaturvedi S, et al. Morphological and Molecular Characterizations of
Psychrophilic Fungus Geomyces destructans from New York Bats with White Nose Syndrome (WNS).
Plos ONE [serial online]. May 2010;5(5):1-12. Available from: Academic Search Complete, Ipswich, MA.
Accessed October 25, 2013.
3. Cornelison, Christopher. Email correspondence. 07 October. 2013.
4. Malik K, Tokkas J, Goyal S. Microbial Pigments: A review. International Journal of Microbial Resource
Technology. December 2012, Vol.1, No.4