This document summarizes an experiment that tested the ability of Streptomyces thermocarboxydus bacteria to reduce hexavalent chromium (CrVI). S. thermocarboxydus was isolated from plant roots and identified using 16sRNA sequencing. The experiment involved three parts: 1) inoculating agar plates and measuring bacterial growth with different concentrations of potassium dichromate, 2) inoculating broth tubes with varying dichromate concentrations and measuring absorbance over time, 3) adding dichromate to bacterial supernatant and measuring absorbance. Results showed that S. thermocarboxydus can reduce CrVI to less toxic CrIII, with its secretions and when growing in concentrations up to 0.5 g/L

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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 bacteria’s 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 Bergey’s 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 3.3:153–160.
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 20:57–60.
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 48:59–68.
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
@575
nm
0.0
g/L
0.02
g/L
0.04
g/L
0.05
g/L
0.06
g/L
0.08
g/L
0
0.05
0.1
0.15
0.2
0.25
0
(w/o
Cr)
0
(w/Cr)
25
48
72
.me
(hrs)
Absorbanc
@575
nm
.5
g/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