1. RESEARCH POSTER PRESENTATION DESIGN 2011
www.PosterPresentations.com Introduction Plate Inoculation Obtain
Materials needed, inoculate plates from Streptomyces
thermocarboxydus culture, and make solution of potassium
dichromate. Titrate to desired concentrations. Soak sterile disks
in concentrations and place in plate in 27 degree Celsius
incubator, observing for 72 hours. Tube Titration Obtain Materials
needed, inoculate tubes from Streptomyces thermocarboxydus culture,
and make solution of potassium dichromate. Titrate to desired
concentrations. Add concentrations to tubes and place in shaking 37
degree Celsius incubator, measuring absorbance every 24 hours for a
72 hour period. Supernatant Absorption Obtain Materials needed and
inoculate TS broth from Streptomyces thermocarboxydus culture.
Place tube in centrifuge and spin at 3000 rpms for 20 mins. Remove
supernatant and make solution, adding 5 micrograms of potassium
dichromate to 20 microliters of inoculated broth. Measure
absorbance and place in shaking 37 degree Celsius incubator,
measuring absorbance every 24 hours for a 72 hour period. Methods
Results Various morphological, metabolic, catalytic, and hydrolytic
tests were done to identify a rhizobacterial species in conjunction
with a 16s rRNA BLAST sequence. We were able to identify our
isolated species as Streptomyces thermocarboxydus. Research was
done on this microbe to identify what properties and information
was known about it. We found an article in which it described the
ability of the microbe to reduce hexavalent chromium. As hexavalent
chromium is a toxic substance to organic matter, this ability
suggests a symbiotic advantage for plants that live among S.
thermocarboxydus. We decided to test the ability of our isolated
organism and see if this ability was present and at what
concentrations this would occur. We designed a three part
experiment to test 1) the toxicity of potassium dichromate to S.
thermocarboxydus, 2) the ability of a growing culture of S.
thermocarboxydus to reduce Cr (VI) at various concentrations, 3)
the ability of secretions from S. thermocarboxydus to reduce an
identified max toxic level of potassium dichromate in solution. The
first part to the experiment produced inconclusive results as no
growth inhibition was able to be detected. Although this may simply
reveal that the species is not susceptible to potassium dichromate,
further experimentation would need to be done to confirm this.
Perhaps the disk method was not the most effective for this species
due to its highly colonial growth pattern. It did not form laws of
bacterial growth, making it difficult to identify areas of
inhibition. Another method to test this would be to pour a series
of agar plates with the potassium dichromate mixed into the media.
The growth ability of the bacteria would then be able to be more
observable. The second part of the experiment provided a little
more data. An increase of absorbance at the 0.06 g/L and 0.08 g/L
concentrations showed that there is some change over time in
absorbance. It also revealed that below 0.06 g/L, the concentration
is so small that change either did not occur or was not observable.
The data was not conclusive however, due to the decrease in
absorbance in the 0.08 g/L samples at 46 hours. It is possible that
this could be due to the presence of bacterial colonies interfering
with spectrophotometer. This experiment revealed that the bacterias
growth was stunted by higher levels of potassium chromate. To
improve this experiment it may be helpful to increase the
concentration of the Cr in the sample. Also, a test to further
understand the reduction of Cr and its detection by absorbance
would be helpful. There may be methods to amplify the detection of
presence of Cr (III) and make readings more accurate. In the third
part of this experiment we tested the ability of the excretions of
the bacteria to reduce the Cr. This experiment was probably the
most conclusive. We discovered that the supernatant may be just as
effective at reducing Cr. However, colonies were found growing in
the culture which does not confirm this. However, there is a
consistent increase in absorption, confirming the reducing ability
of the bacteria. Throughout all of our experiments we were able to
isolate and identify the bacterium S. thermocarboxydus. Although
our experiments were not conclusive, we were able to confirm that
the bacterial species reduces chromium, and that it can survive in
concentrations up to 0.5 g/L. An understanding of this process was
determined and can be used to further investigate this reducing
ability. This knowledge could be further used in environmental
purposes. If this species is effective in reducing the toxic
substance, it could be used to remove the chemical from industrial
spill and leakages. We can understand its role in plant symbiosis
in protecting the organism from toxic levels of Cr (VI). Discussion
Conclusions 16sRNA Sequencing To identify the microbe that was
isolated, the 16sRNA sequence that was run through PCR was sent in
and sequenced and then analyzed. The analysis and comparison of
this sequence through NCIB produced a list of several Streptomyces
sp. but the top result was S. thermocarboxydus with a 99% match.
Through comparing the identifying test results that we retrieved
with those found in Bergeys Manual, we were able to confidently say
it matched the species that was identified with the sequence.
Chromium reduction In the Cr experiment that used the Kirby-Bauer
technique, there was not any conclusive data produced. After 72
hours of incubation, the bacteria produced a significant coverage
of colonies across the plates. We expected the quadrants with Cr
disks of higher concentration to have less growth; however, it
appeared to have slightly more growth. This was not consistent
either as our control had more growth than the quadrant of low Cr
concentration but less than the quadrant of high concentration.
Perhaps the experiment could be repeated with disks of higher
concentration to produce more significant results. In Graph 1 it
displays the results of the experiment in which S. thermocarboxydus
was grown in a solution of TS broth and various concentrations of
potassium dichromate over 46 hours. The data reveals various
results. It appears that any concentration less than .06 g/L of Cr
is not significant enough to detect reduction. At the concentration
0.06 g/L there is a positive trend in absorbance. However at the
concentration 0.08 g/L the absorbance decreases at 46 hours. This
may be an experimental error due to the presence of bacteria in the
solution interfering with the absorbance readings. Bacterial
colonies could be observed growing in decreasing amounts with an
increase in Cr concentration. After 46 hours, samples with Cr
concentrations of 0.0 g/L and 0.02 g/L were too dense in bacterial
growth to acquire a reading. The third part of the experiment
tested the reducing ability of the supernatant (secretions) of the
bacterial culture. Graph 2 reveals these results. This experiment
produced a linear increase in absorbance. This shows that there was
a consistent reduction of Cr over the 72 hours. Some bacteria were
discovered in the bottom of the tube after 48 hours. This
experiment also showed that S. thermocarboxydus can tolerate as
much as .5 g/L of potassium dichromate. Sources Desjardin V, Bayard
R, Lejeune P, Gourdon R. 2003. Utilisation of Supernatants of Pure
Cultures of Streptomyces Thermocarboxydus NH50 to Reduce Chromium
Toxicity and Mobility in Contaminated Soils. Water, Air & Soil
Pollution: Focus 3.3:153160. Kawakami H, Inuzuka H, Mochizuki K,
Muto T, Ohkusu K, Yaguchi T, Yamagishi Y, Mikamo H. 2014. Case of
keratitis caused by Streptomyces thermocarboxydus. Journal of
Infection and Chemotherapy 20:5760. Kim SB, Falconer C., Williams
E., Goodfellow M. 1998. Streptomyces thermocarboxydovorans sp. nov.
and Streptomyces thermocarboxydus sp. nov., two moderately
thermophilic carboxydotrophic species from soil. International
Journal of Systematic Bacteriology 48:5968. Characterization of
Streptomyces thermocarboxydus Growth and Chromium VI Reduction in a
Rhizomic Environment Josh McAlister and Chad Schlagel The purpose
of this experiment was to test the mobility and reduction of
Chromium VI in the presence of S. thermocarboxydus in a riparian
environment. This bacteria was isolated from the roots of Coreopsis
lancelota through a dilution technique. Multiple microbiological
survey tests, including a blast sequence of 16sRNA, were performed
to identify the correct bacteria that had been isolated. This blast
was compared, and matched the genome with 99 % accuracy. S.
thermocarboxydus is a moderately thermophilic, gram negative,
facultatively chemolithotrophic actinomycete. It is found in the
riparian environment and is carboxydotrophic. It oxidizes carbon
monoxide and hydrogen, giving the plant the nutrients needed for
growth. In addition, it reduces chromium VI to chromium III,
allowing the plant to have an optimal growth environment. 0 0.01
0.02 0.03 0.04 0.05 0.06 0 24 46 .me(hrs) Absorbance@575nm 0.0g/L
0.02g/L 0.04g/L 0.05g/L 0.06g/L 0.08g/L 0 0.05 0.1 0.15 0.2 0.25
0(w/oCr) 0(w/Cr) 25 48 72 .me(hrs) Absorbanc@575nm .5g/L Graph 1:
The change in absorbance at 575 nm for samples containing S.
thermocarboxydus over time. The amount of Cr (VI) that live active
cultures of S. thermocarboxydus reduces is observed by the increase
in absorbance at 575 nm. Different concentrations were observed
over 46 hours to determine the ability and susceptibility of the
bacteria to reduce the chromium. Graph 2: The change in absorbance
at 575 nm for a sample containing S. thermocarboxydus supernatant
over time. The amount of Cr (VI) that is able to be reduced in the
supernatant of S. thermocarboxydus is observed by the increase in
absorbance at 575 nm. A single concentration of 0.5 mg/L was tested
and shows an increase in absorption (increase in Cr III) after 72
hours. Table 1: Bacteria Morphology and Microbial Test Results
Figure 1: Plate Inoculation, Tube Titration, and Potassium
Dichromate Figure 2: Results of Tube Titration Experiment
