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Optimizing the Freshwater Denitrification Process of Heterotrophic Bacteria Using Iron
Nanoparticles
Katie Haske
With Mercedes Dick, Ashley Hollinshead, and Carolyn Mazzuca
CHE 480
Abstract
Urgency to remove contaminants from the environment is becoming dire as we discover how
harmful each is to life sustainability. Nitrate is a health-endangering carcinogen, and is therefore
the focus of our experimentation. This research group tested the effectiveness of Pseudomonas
aeruginosa, in conjunction with zero-valent iron nanoparticles, at reducing nitrate in water by
converting it to ammonia and elemental hydrogen gas. The nanoparticles were created through
the impregnation of iron salt solutions into environmentally-friendly milo seed carbon black.
After these samples underwent Carbothermal reduction, each was combined with cultured P.
aeruginosa and a 10mM nitrate solution and left to react for 46 hours. Samples were run in
triplicates at 12°C, 25°C, and 35°C, and repeated at 35°C with initial nitrate solution
concentrations of 0mM, 50mM, and 100mM NO3. The results yielded no significant change in
nitrate or ammonia concentration between the various temperatures and initial NO3
concentrations.
2
Introduction
Nitrate is a known carcinogen that is common to freshwater systems due to agricultural
and industrial runoff (Shin & Cha 2008). Lives and environments worldwide, including the
Emiquon Nature Reserve in Illinois, are being negatively impacted by this toxin as populations
require more farmland and fresh water to sustain life. Though health problems have not been
decisively proven, correlations have been found by the EPA resulting the nitrate concentration in
drinking water required to be 10mg1-1
NO3
-
-N or below (Shin & Cha 2008). Various means of
reducing nitrate concentration in water have been used, such as ion exchange and reverse
osmosis. However, each of these have posed economical complications due to operational costs
and/or waste removal issues (Shin & Cha 2008).
An alternative water treatment process to the previously mentioned cost-ineffective and
waste-abundant choices is using agent zero-valent iron (ZVI). ZVI has been proven to be a
powerful reducing agent of pollutants, but inevitable drawbacks exist. The equation below
displays how the iron works:
NO3
-
+ 4Fe(0) +10H+
→ 4Fe2+
+ NH4
+
+ 2H2O
The ZVI reduces the nitrate into ammonia, which in high concentrations can also be dangerous to
biological systems. Though the process requires acidic conditions and introduces excess
ammonia into water systems, it is more cost-effective and green than existing processes,
especially when the ZVI is produced from carbon black made from sustainable milo seed (Bapat,
Manahan &Larsen 1999).
Biological denitrification, executed with Pseudomonas aeruginosa, is an additional
process option in reducing the nitrate concentration of freshwater systems. These bacteria are
hardy and can survive under various, “hostile conditions” (Stoodley, Costerton & Stoodley
3
2004). With this choice, the nitrate is reduced to harmless elemental nitrogen, instead of
ammonia which can accumulate and interrupt biological systems (Shin & Cha 2008). However,
the unnatural addition of biological material will still impact and alter the nature of the area
undergoing treatment. P. aerurinosa has been known to be cause dangerous infections in
humans, so it must be handled with care and in careful concentrations (Hauser & Rello 2003).
This process, like ion change and reverse osmosis, results in denitrification, as well as an
increase in unwelcome materials into the water system—infectious bacteria and nanoparticles—
requiring the system to undergo further treatment (Klaper).
In a study by Shin and Cha, zero-valent iron works more effectively in a nanoparticle
form. The size introduces numerous benefits in the process such as increased surface area and
larger versatility in application location. The zero-valent iron nanoparticles (NZVI), in
conjunction with nitrate reducing culture, are expected to work as displayed in the following
reaction:
2NO3
-
+ 5H2 → N2 + 4H2O + 2OH-
Rather than reducing the nitrate into ammonia, it can be reduced to innocuous nitrogen gas with
the use of biotic reduction. The bacteria use the electrons from the NZVI to make this reaction
occur (Park & Yoo 2009) . The path to this type of denitrification begins by creating sustainable
BioChar.
Carbon, in it’s activated, porous form, is an effective component in different processes of
environment purification. It is a compelling component in decontaminating water, air, and other
materials. Coal, wood, lignite, and agricultural wastes are only a few raw materials that can be
used to create activated carbons (Bapat, Manahan &Larsen 1999). Because this material is so
commonly used in decontamination processes, deactivated, used carbon itself becomes a
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contaminant because of its abundance. From an economic and environmental position,
reactivating spent carbon has become crucial in order to reduce problems associated with
transportation of, disposal of, and cost of spent activated carbon (McGowin 1991).
This experiment utilizes the ChemChar reverse-burn gasification of Milo seed (sorghum
vulgare) to return deactivated carbon to its favorable, porous identity (McGowin, Kinner &
Manahan 1991). Milo seed is a green, biological alternative used for activated carbon production,
a component significantly less harmful to the environment than typically-used charcoal. The
reactivation process consists of three thermal steps: 1. water and volatile adsorbates vaporization
at 200°C, 2. additional volatile adsorbate vaporization and decomposition at 200°C-500°C, and
3. remaining adsorbate pyrolysis and activated char production at 500°C-700°C (McGowin
1991). The resulting carbon, if conducted successfully, will be once again useful in material
purification due to its returned high-surface area, high-absorptive identity (Bapat, Manahan
&Larsen 1999).
This activated carbon is used to produce the NZVI. The method is simple: react soluble
iron salts with carbon black, or thrice gasified milo Biochar, as described above. Two iron salt
solutions, ferric nitrate and iron (III) citrate, were created, combined with the char, and placed
through a Carbothermal reduction to produce the reactive and highly sorptive zero-valent iron
nanoparticles (NZVI) that can remediate organic compounds that it comes into contact with
(Hoch, et al. 2008). In conjunction with the selected bacteria, P. aeruginosa, nitrate
concentration in water is expected to be reduced.
The intention of this experiment determine the optimal conditions that bacteria denitrifies
in conjunction with NZVI. Temperature conditions were varied to simulate real-world
temperatures. The anaerobic process will be tested at 12°C, 25°C, and 35°C, representing
5
potential weather conditions. A second experiment was conducted at 35°C to observe how
varying initial nitrate concentrations with 0mM, 50mM and 100mM solutions affects final nitrate
reduction and ammonia production.
Experimentation
ChemChar Gasification Process
Milo seed was baked in 9”x13” aluminum trays for at least 24 hours at 160°C. The
bottoms of the trays were evenly covered in about a 1” thick layer of seeds, with all non-milo
components removed. The baked tray of milo was removed, stirred, redistributed evenly along
the bottom, and then returned into the oven at 220°C for an additional minimum of 24 hours. The
twice-baked milo seed was then stored until ready to execute the ChemChar gasification process.
To set up the ChemChar apparatus, a hollow iron tube was mounted in a vertical position.
Depending on the amount of baked milo seed, this tube was occasionally substituted with a
thinner Pyrex tube to ensure that the milo could reach the top of the tube when filled. 2-3 inches
of fiberglass insulation was pushed into the bottom of the iron tube followed by a rubber stopper
with a glass tube in the center (to eventually be attached to the compressed oxygen tank). Once
this was set-up, baked milo seed was poured into the tube until it reached about 1 inch from the
top. If there was not enough milo to fill the tube to the top, the thinner glass tube was used. It
was best to pour the seed through a wide-necked funnel so that large chunks could be crushed
with a spatula before entering the tube. The more uniformly sized the material, the more evenly it
burned. As the seed was added, the column was packed by gently tapping it with a hand to
compress it evenly. With rubber tubing, the glass tube in the stopper was attached to the
compressed oxygen tank.
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The compressed oxygen tank was then turned on enough to establish a gentle flow
through the iron tube. When the oxygen was too high, milo seed was blown off the top; when the
flow was too low, the milo could not sustain a flame front. When appropriate flow was
established, the top layer was ignited with a small propane torch. Again, the oxygen was adjusted
so that the flame front was a small, glowing simmer. The speed of the flame was tracked by
spraying deionized water on the side of the iron tube. The position at which the water evaporated
immediately revealed how far along in the iron tube the flame front was. Once it had reached the
location of the fiberglass insulation, the oxygen was turned off, quenching the flame. This
concluded the first-gasification of the milo seed.
To gasify the milo seed a second time, the ChemChar apparatus was constructed
identically to the first gasification, but instead the vertical tube was filled with the once-gasified
milo seed; rather than immediately pouring the once-gasified milo into the iron tube, the milo
was weighed, and 5% by weight of nanopure water was mixed into the seed. The water was
poured on top of the milo and mixed in with a spatula carefully as to not to crush the milo. At
this point, the seed was transferred into the iron tube and heated analogously to the first-
gasification process. The third gasification was executed similarly, but with a 15% by weight
addition of nanopure water. This thrice gasified BioChar was then stored until it was used in the
impregnation process.
Zero-Valent Iron Nanoparticle Synthesis
Two iron solution were made: 2.6mM ferric nitrate solution and another with a 1.5mM
iron (III) citrate solution. Each was then capped with Parafilm and inverted repeatedly until the
salts were completely dissolved into the solution. Next, the BioChar was prepared.
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The thrice-gasified BioChar was grinded with a mortar and pestle until the char was a
fine powder. 6.25g of this ground BioChar was added to a 500 mL purged glass jar, as well as
the ferric nitrate solution. In the same fashion, 5.12g of ground BioChar was added into a second
purged 500 mL glass jar with the iron (III) citrate solution. Each of these jars were then placed
into an automatic shaker at 200RPM for 24 hours.
Once the time was up with the shaker, the solutions were put through separate vacuum
filtration systems. To set up this apparatus, a rubber stopper was placed into the opening of a 250
mL side-arm flask, and then a Buchner funnel was placed inside that. An appropriately sized
filter paper was put inside the funnel and secured down with a small amount of corresponding
iron salt solution. The side arm of the flask was connected to the vacuum system with a rubber
tube. The vacuum was turned on enough to create a gentle suction. Little by little, the iron
solutions were poured into the vacuum filtration system. Rotating the jar while pouring had
helped ensure that all of the impregnated BioChar was transferred into the funnel since it often
adhered to the sides. This process was identical for both solutions. When the solid was dried, it
was transferred into a storage container (after scraping as much off the filter paper as possible)
after recording the weight for the yielded product.
Carbothermal Reduction of Iron Salts
Materials necessary for this process were a tube furnace, an asbestos tube, compressed
argon tank, two rubber stoppers, a compressed gas regulator, a mineral oil bubbler, two ceramic
boats, two glass jars with lids, and a spatula.
Each of the dry iron salt solutions collected above were transferred into separate ceramic
boats until the entire bottom of each respective boat was covered completely by an even layer of
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salt. The boats were then slid into the center of the asbestos tube so that they were side by side.
This tube containing the ceramic boats was then returned to the tube furnace, each side was
plugged with a high-vacuum greased rubber stopper with a glass rod in the center. One stopper
was connected to the compressed argon tank and the other was attached to the mineral oil
bubbler, each with rubber tubing. At this point, the gas tank was turned on slowly—enough to
create a gentle bubble in the bubbler. The gentle bubble, which was found by watching the
bubble pace in the mineral oil bubbler, was ideal to prevent the waste of argon gas with equal
effectiveness of removing oxygen and creating positive pressure inside the asbestos tube.
The apparatus was run under these conditions for one hour, and then the heat was turned
on to 800°C without ceasing the flow of argon gas. With temperature high, the apparatus was left
to heat for six hours. After this time was complete, the heat was shut off and the furnace was
allowed to cool for approximately one hour. The samples were then stored in glass jars that had
been purged for future use.
X-ray diffraction followed this process to test the success of the Carbothermal reduction
in producing zero-valent iron nanoparticles. The reduced iron samples were ground with an agate
mortar and pestle until each was a fine powder, so that it can be used in powder x-ray diffraction
(XRD). A small aluminum XRD disc with about a 1” diameter was covered on one side with
double stick tape. The edges of the tape were trimmed with a razor blade. A thin layer of iron
powder was spread onto the tape. This disc was then placed into the diffractor at 2° per minute
until 100°. With successful detection of the iron through this process, it would be evident that
NZVI was present in the samples and the experimentation could proceed.
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Culturing Bacteria
Before the culturing the bacteria, LB broth was created for the bacteria to develop in. LB
broth is a nutrient rich broth that encouraged high levels of growth; however, potential nitrate in
the broth likely skewed the final results, so a nitrate free broth was necessary.
To create the nitrate-free broth, the following components were dissolved in 1L of
nanopure water:
1.   Dextrose: 0.860g
2.   HEPES acid: 14.227g
3.   HEPES sodium salt: 7.8189g
4.   KH2PO4: 0.2982g
5.   MnSO4*H2O: 0.1001g
6.   FeCl2*4H2O: 0.4505g
7.   CaCl*2H2O: 0.0250g
8.   CoCl2*2H2O: 0.1902g
To this solution, 1mL of second 1L solution was added. The second 1L solution was composed
of the following components dissolved into 1L of nanopure water:
1.   CuCl2*2H2O: 0.0302g
2.   H3BO3: 0.0309g
3.   ZnCl2: 0.0525g
4.   MgCl2*6H2O: 0.1000g
5.   NiCl2: 0.0239g
6.   MnNa2O4: 0.0371g
Once this nitrate-free broth was complete, 0.500M HCl was added until the pH was brought to 7.
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5mL of each broth was put into several 18mm sterile glass test tubes. Bacteria was
inoculated into six tubes of this broth once a week to ensure the bacteria is healthy and active. To
accomplish this, the opening of a test tube and a small metal loop first were flamed in a Bunson
burner for sterilization. The loop was then dipped into a supplied sample of bacteria (inoculated
in LB broth) and transferred into a test tube with nitrate-free broth. The test tubes were promptly
recapped and the metal loop sterilized in the Bunsen burner flame. This process was repeated
every week with six test tubes of new nitrate-free broth. Once transferred, the test tubes were
placed into a 35°C incubator for 24 hours and then into a refrigerator until needed, as
recommended by previous literature (Williams, Rowe, Romero & Eagon 1978).
Creating Solutions for Experiment 1 and Experiment 2
For the trials conducted at varying temperatures, experiment 1, 10mM nitrate solution
was created from solid KNO3 and nanopure water.
For experiment 2, solutions of 0mM, 50mM, and 100mM were created from KNO3 and
nanopure water.
Anaerobic Biotic Nitrate Reduction Process
For experiment 1, there were 3 trials and a control—one Wheaton jar per sample. To each
of these jars, 6mL of the nitrate solution was added, 54mL of nitrate-free broth, which were
measured in appropriately-sized graduated cylinders, and 0.30g of iron nanoparticles.
Because of the danger of the bacteria, the following steps were executed in a Laminar
flow hood. Five test tubes of bacteria-filled nitrate-free broth were poured and mixed into a 250
mL Erlenmeyer flask until the solution was uniform. With a 5 mL plastic sterile volumetric
11
pipet, 5 mL of bacteria solution was added into the Wheaton jars containing samples 1, 2, and 3.
An additional 3mL of of bacteria solution was pipetted into a scintillation vial to test for bacteria
presences with the DAPI process later.
Time zero-samples were taken with autoclaved 12-inch long needles attached to sterile
syringes to draw up four 20 mL samples, one from each Wheaton jar, and dispense each into
individual whirl-packs. These were then stored in the freezer for future analysis.
Next was the process for collecting time-final samples. Small syringes that were stuffed
with cotton (to vent the solutions without contaminating the air with bacteria) were pierced
through the top of each Wheaton jar. The previously used 12-inch needles were made sure to be
submerged into the solutions, and then connected to tubing that was connected to a nitrogen gas
tank. The jars were purged for 15 minutes with nitrogen gas. When this time was complete, both
the 12-inch needles and the small syringes were removed, the gas was shut off, and the holes in
the lids were sealed with grease. These jars were then put into a shaker for 46 more hours at
200RPM, and then the solutions were transferred to desterilized falcon tubes for later analysis.
This process was repeated for each temperature trial (12, 25, and 35°C) in experiment 1,
as well as for all the trials in experiment 2; however, 11 jars underwent this process in
experiment 2 rather than four. Three concentrations were tested: 0mM NO3, 50mM NO3, and
100mM NO3. Each concentration consisted of 3 replicates (composed of nitrate solution,
nanoparticles, and bacteria) and 1 negative control (composed of solution and nanoparticles).
Additionally, 3 positive controls (composed of nanopure water, nanoparticles, and bacteria) were
tested.
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Phenate Method for Ammonia Concentration Determination
To determine how much ammonia was produced over the course of this experiment, a
series of solutions were made to be run through a spectrophotometer to test for absorbance
(Barletti, Paver, & Dungey 2007). The following solutions were made:
1.   Phenol/Ethanol: Dilute 5.55 mL phenol in a 50 mL volumetric flask with 95% ethanol
2.   Sodium nitroprusside solution 0.5%: Dissolve 0.5 g of sodium nitroprusside in 100
mL of nanopure water
3.   Alkaline citrate: Dissolve 200 g of trisodium citrate and 10 g of sodium hydroxide in
1 L of nanopure water in a 1L volumetric flask.
4.   Sodium hypochlorite 5-6%: Use commercial bleach
5.   Oxidizing solution: Mix 100 mL of alkaline citrate solution with 25 mL of sodium
hypochlorite solution
6.   Stock ammonia solution: Dissolve 3.819 g of anhydrous ammonium chloride (dried at
105° C) in 1 L of nanopure water
Twenty 20 mL scintillation vials were then labeled appropriately. The following 7 standards
were made using an ammonia working stock solution and put into scintillation vials.
Working stock solution: 0.10 mL of stock ammonia solution was added to 100 mL
volumetric flask and bring to volume with nanopure water
Blank: 15 mL of nanopure water
1.   15 mL of working stock solution
2.   Add 7.5 mL of working stock solution to 7.5 mL of nanopure water
3.   Add 3 mL of working stock solution to 12 mL of nanopure water
4.   Add 1.5 mL of working stock solution to 13.5 mL of nanopure water
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5.   Add 750µL of working stock solution to 14.25 mL of nanopure water
6.   Add 375µL of working stock solution to 14.625 mL of nanopure water
7.   Add 150µL of working stock solution to 14.85 mL of nanopure water
In addition to the standards, solutions with the samples were made. With a 15 mL disposable
serological pipette, 15 mL of each sample at To and Tf was pipetted into respective scintillation
vials. 600µL of sodium nitroprusside solution was pipetted into each vial (both the vials with the
standards and the vials with the samples) using an automatic micropipettor. 1.5 mL of the
oxidizing solution was then added to all the scintillation vials, as well. Once all solutions and
standards were complete, the vials were capped, inverted three times, and covered completely
with aluminum foil to protect the solutions from light. The covered vials were left to sit for a
minimum of one hour. When the setting time was complete, the absorbance’s of all vials were
measured using a Spec 20 Geneysis spectrophotometer at 640nm. This process was repeated for
the samples collected from experiment 2.
Ion Chromatography Method for Nitrate Concentration Determination
Ion Chromatography was used to determine the nitrate concentration of the time initial
and time final samples (Dungey 2013). Standard solutions were created using ion
chromatography standard 6. In IC Vials, standards were diluted with 5, 50, 100, 500, and
2000µL up to 5mL with nanopure water in IC vials. 50µL of each sample, time-initial and time-
final, collected was diluted up to 5mL with nanopure water into IC vials. Each vial was placed
into the chromatograph for analysis. This process was repeated for the samples collected from
experiment 2.
14
DAPI Protocol for Bacteria Quantification
The DAPI protocol is a procedure used to quantify the bacteria in samples (Dungey
2016). The 3mL bacteria sample preserved from the Anaerobic Biotic Nitrate Reduction Process
was used in this process. To set up this procedure, a 25mm diameter fritted glass filter was
secured onto the neck of a 250mL side arm flask with plastic clamps. A 0.22𝜇m pore backing
filter was placed on the glass filter, followed by a 0.22𝜇m pore, black membrane filter, and
finally a second fritted glass filter. With rubber tubing, this system was attached to a vacuum at
5PSI. The following materials were added to the funnel in numbered order:
1.   1mL nanopure water
2.   Sample
a.   LB broth samples were diluted to 10𝜇M with saline
b.   Nitrate-free broth sample was diluted to 100𝜇M
3.   200𝜇L DAPI
With the vacuum still on and these components added to the filter, the system was covered with
aluminum to block out light and left to react for 5 minutes.
Once this was complete, the set-up was disassembled, and the membrane filter was
placed onto a microscope slide overtop a drop of FF immersion oil. A second drop of oil was
placed on top of the filter, followed by the slide cover. With a Kimwipe, the cover was gently
pressed down, flattening the layers between the glass. The slide was then viewed under a
microscope.
15
Results
Figure 1. Powder X-Ray Diffraction of iron nitrate sample.
Sample was at 2 degrees per minute at 2.29 angstroms. Peak at 69°C identifies successful
production of iron nitrate nanoparticles following nanoparticle impregnation of Biochar.
Temperature Sample
Absolute
Difference (mg/L) Percent
Difference
Average Percent
Difference +/- 1
SD (without
control)
12°C
Control -0.8887 -45.3
-14 +/- 101 -0.3716 -28.9
2 -0.0437 -4.1
3 -0.1002 -8.6
25°C
Control +0.3129 +36.1
-22 +/- 141 -0.8359 -41.1
2 -0.1801 -13.7
3 -0.1299 -10.0
35°C
Control -0.0113 -1.1
-4 +/- 51 -0.0238 -2.5
2 +0.0029 +0.3
3 -0.1033 -10.2
Table 1. Nitrate Change Results
The nitrate concentration change results, from time initial (T0) to time final (TF) collected
through ion chromatography. 5 samples of varying concentrations of IC standard 6, and of each
control and sample diluted from 50µL to 5µL were collected analyzed with Dionex ICS 2000.
16
Absolute difference in concentration was calculated by subtracting T0 raw value from T0 raw
value. Percent difference was calculated by subtracting TF from T0, dividing the difference by T0,
and multiplying the value by 100%. The standard deviation was calculated excluding the control.
Nitrate
10 20 30
Temperature (°C)
Figure 2. Percent Change in Nitrate Concentration
The blue circles represent the samples, the pink triangles represent the control (samples lacking
Pseudomonas aeruginosa), the error bars are ± one standard deviation, and the gray bars are the
average percent change in concentration all with respect to temperature groups.
-­‐50
-­‐40
-­‐30
-­‐20
-­‐10
0
10
20
30
40
50
0 10 20 30 40
PercentChange(%)
17
Temperature Sample Absolute
Difference in
Concentration
(mg/L)
Percent
Difference in
Concentration
Average Percent
Difference +/- 1
SD (without
control)
12°C
Control +0.0008 -2.89
23 +/- 101 -0.0100 +20.5
2 -0.0179 +33.0
3 -0.0163 +40.3
25°C
Control -0.0109 +41.1
12 +/- 851 -0.0161 +100.
2 +0.0133 -53.9
3 +0.0038 -38.1
37°C
Control +0.0009 -6.85
-54 +/- 341 -0.0121 +36.3
2 -0.0215 +53.7
3 -0.0181 +73.4
Table 2. Ammonia Change Results
The ammonia absorbance change results, from time initial (T0) to time final (TF) collected
through the phenate method and run through a spectrophotometer. Absolute difference in
absorbance was calculated by subtracting T0 raw value from TF raw value. Percent difference
was calculated by subtracting TF from T0, dividing the difference by T0, and multiplying the value
by 100%. The standard deviation was calculated excluding the control.
18
Ammonia
Figure 3. Percent Change in Ammonia Concentration
The blue circles represent the trials, the pink triangles represent the control, the error bars are ±
one standard deviation, and the gray bars are the average percent change in concentration all with
respect to temperature groups.
-­‐175
-­‐150
-­‐125
-­‐100
-­‐75
-­‐50
-­‐25
0
25
50
75
0 5 10 15 20 25 30 35 40
PercentChange(%)
Temperature (oC)
19
Nitrate
Concentration of
Initial Solution
(mg/L)
Sample
Absolute Change in
Concentration
(mg/L)
Percent
Change in
Concentration
(mg/L)
Average
Percent
Difference +/-
1 SD
Positive Control 1 0.0033 -132
-219 +/- 980 Positive Control 2 0.0056 -200
Positive Control 3 0.0052 -325
Negative Control -0.0804 1.21
-3 +/- 5
50 1 0.1478 -2.52
2 0.4287 -7.84
3 -0.0857 1.54
Negative Control -0.2701 2.14
1 +/- 8
100 1 -0.5638 4.70
2 0.9102 -8.00
3 -0.7826 6.55
Table 3. Trial 2 Nitrate Change Results
Trial 2 was run at 35°C to maximize resulting affect of initial nitrate concentration on final
nitrate concentration. Positive control consisted of nanopure water, NZVI, and bacteria, negative
control consisted of solution and NZVI, and the replicated consisted of solution, NZVI, and
bacteria.
20
Figure 4. Trial 2 Percent Change in Nitrate Concentration
The blue dots represent the replicates per concentration trial, the pink dots represent the negative
control per respective trial, the grey bars represent the average, and the pink triangles represent
the control.
Nitrate
Concentration of
Initial Solution
(mg/L)
Sample Absolute Change
in Concentration
(mg/L)
Percent
Change in
Concentration
(mg/L)
Average
Percent
Difference
+/- 1 SD
0
Positive Control 1
0 0 9 +/- 9
Positive Control 2
-0.0033 +10.4
Positive Control 3 -0.0065 +17.2
50
Negative Control -0.0131 +31.6
22 +/- 71 -0.0065 +15.5
2 -0.0115 +23.9
3 -0.0147 +28.3
100
Negative Control -0.0106 +23.4
35 +/- 201 -0.0131 +27.3
2 -0.0090 +19.8
3 -0.0303 +58.2
Table 4. Trial 2 Ammonia Change Results
-­‐350
-­‐300
-­‐250
-­‐200
-­‐150
-­‐100
-­‐50
0
50
-­‐20 0 20 40 60 80 100 120
Percent	
  Change	
  (%)
Initial	
  Nitrate	
  Concentration	
  (mg/L)
Percent	
  Change	
  in	
  Nitrate
21
Trial 2 was run at 35°C to maximize resulting affect of initial nitrate concentration on final
nitrate concentration. Positive control consisted of nanopure water, NZVI, and bacteria, negative
control consisted of solution and NZVI, and the replicated consisted of solution, NZVI, and
bacteria.
Figure 5. Trial 2 Percent Change in Ammonia
The blue dots represent the replicates per concentration trial, the pink dots represent the negative
control per respective trial, the grey bars represent the average, and the pink triangles represent
the control.
-­‐10
0
10
20
30
40
50
60
70
-­‐20 0 20 40 60 80 100 120
Percent	
  Change	
  (%)
Initial	
  Nitrate	
  Concentration	
  (mg/L)
Percent	
  Change	
  in	
  Ammonia
22
Figure 6. Bacteria Quantification
Images of Pseudomonas aeruginosa taken through optical lens of microscope available through
the DAPI protocol. The left image displays illuminated bacteria grown in LB broth, and the left
image displays illuminated bacteria grown in low-nutrient nitrate-free broth.
Discussion
Figure 1 displays the powder x-ray diffraction results of the iron nitrate samples. The
peak just below 70°C indicates the successful production of zero-valent iron nanoparticles. With
the existence of NZVI in the samples, it was predicted that nitrate will be reduced, with or
without additional biological denitrification. The supplemental biological component, P.
aeruginosa is displayed in Figure 4. Through the DAPI process, existing bacteria in the samples
were clearly illuminated under a microscope. The bacteria cultured in the LB broth was thriving
as opposed the that cultured into the nitrate-free broth—the broth used throughout
experimentation. This broth was made of various components and only a small amount dextrose
that the bacteria could survive from; it was likely that the bacteria was starved. Additionally to
the DAPI process to determine existence of bacteria, Anna Ball, biology student at the University
of Illinois at Springfield, did gram stains of nitrate-free broth cultures confirming the lack of
23
active or inactive bacteria in the solutions. The samples relied solely on the NZVI for nitrate
reduction and ammonia production.
The percent change in nitrate concentration (see Table 1 and Figure 2) of the triplicate
samples and the respective controls behaved oppositely. At 12°C, the control reduced the nitrate
significantly more than each of the samples, opposite that of the samples ran at 25°C. The
control was within the narrow sample array of 35°C, clustered around 0% change. The overall
error range for the change in nitrate concentration was consistent with zero percent change. This
suggests that temperature has no effect on the reduction of nitrate in 100mM nitrate solutions
over 46 hours. This could be due possibly to the lack of bacteria, time that samples were allowed
to react, or the concentration of the nitrate solutions that the nanoparticles and bacteria were
working in.
Table 2 and Figure 3 display the percent change in the ammonia concentration over the
course of the experiment. Though the control of each temperature trial was above the average of
the corresponding sample averages, opposite of what was expected, the range and behavior of the
samples made this trend unreliable to conclude from (Shin & Cha 2008). The controls of 12°C
and 35°C were significantly larger and of opposite sign of the corresponding triplicate samples.
Though the control of the 25°C trial is close to average of the samples, the span of percent
change ranges over 150% with two of the samples falling beyond the error bars, double that of
both the other trials. 4 out of the 9 samples and 2 of the 3 controls were placed beyond the error
bars of the respective temperature. This was predicted to be the results of inappropriate initial
nitrate concentration selection that the nanoparticles and bacteria were reacting in.
A final trial experimental section (see Table 3 and Figure 4) with varying initial nitrate
solution concentrations was conducted to quantify how much the change in nitrate and ammonia
24
was affected by this concentration. Three concentrations were tested: 0mM NO3, 50mM No3,
and 100mM NO3. Each concentration consisted of 3 replicates (nitrate solution, nanoparticles,
and bacteria) and 1 negative control (solution and nanoparticles). Additionally, 3 positive
controls (nanopure water, nanoparticles, and bacteria) were tested. The nitrate percent change of
the positive controls varied greatly from the other concentrations. Even though this solution had
a nitrate concentration of 0mM, the average percent difference of nitrate was -219%, compared
to the nitrate solutions having average percent differences of -2.9% and 1.1%. Additionally, the
nitrate solutions behaved oppositely: the 50mM solution had a decrease in nitrate concentration
and the 100mM solution had an increase in nitrate solution. This trial suggests that nitrate
concentration has no effect on nitrate reduction.
The percent change in the ammonia for each concentration mostly clustered closely;
however, there is no significant difference across trials. The positive controls had a smaller
increase in ammonia that the other solutions, as expected. However, the negative controls
without bacteria behaved so similarly to the replicates with bacteria that the impact of the
bacteria appeared to be negligible, further confirming the lack of bacteria in the solutions as
displayed in Figure 4. Initial nitrate concentration has no effect on how much ammonia is
produced or reduced.
Conclusion
The purpose of this experiment was to define optimal conditions that zero-valent iron
nanoparticles in conjunction with the bacteria Pseudomonas aeruginosa could denitrify water
systems more efficiently with the intention of eventually applying it to the Emiquon Nature
Preserve in Illinois. NZVI was successfully created through the various processes described, but
25
particles were not as effective as expected. The affect of bacteria on this system is inconclusive
since did not survive through the trials.
Neither temperature nor initial nitrate concentration were proven to affect how the
concentration of the nitrate or the ammonia changed after anaerobic exposure to NZVI and
bacteria. However, future research will benefit from this project.
A different type of bacteria, such as an active sludge, is recommended. An already active
denitrifying biological material is likely to reduced nitrate concentration in water systems with
the addition of NZVI. This will eliminate many of the suspected errors that existed throughout
this project and could be the solution to denitrifying contaminated freshwater systems.
26
References
Bapat H, Manahan S, Larsen D. 1999. An activated carbon product prepared from milo (sorghum
vulgare) grain for use in hazardous waste gasification by chemchar cocurrent flow
gasification. Chemosphere.39(1): 23-33.
Bartletti J, Paver S, Dungey K. 2007. Standard operating procedure: phenate method. 1-25.
Dungey K. 2013. Standard operating procedure: ion chromatography. 1-12
Dungey K. 2016. Standard operating procedure: determination of bacterial counts. 1-2.
Hauser A, Rello J. 2003. Severe infections caused by pseudomonas aeruginosa. New York:
Springer Science.
Hoch, et al. 2008. Carbothermal synthesis of carbon-supported nanoscale zero-valent ion
particles for the remediation of hexavalent chromium. 2600-2605.
Klaper R. Environmental implications of nanotechnology: developing sustainable
nanotechnology.
McGowin A, Kinner, L, Manahan S. 1991. Chemchar process for carbon re-Activation.
Chemosphere. 22(12): 1191-1209.
Park J, Yoo Y. 2009. Biological nitrate removal in industrial wastewater treatment: which
electron donor we can choose. Microbiology Technology. 82:415-429.
Shin K-H, Cha DK. 2008. Microbial reduction of nitrate in the presence of nanoscale zero-valent
iron. Chemosphere. 72(2): 257-262.
Stoodley L, Costerton W, Stoodley P. 2004. Bacterial biofilms: from the natural environment to
infectious diseases. Nature Reviews Microbiology. 2: 95-108.
Williams D, Rowe J, Romero P, Eagon R. 1978. Denitrifying pseudomonas aeruginosa: some
parameters of growth and active transport. Applied and Environmental Microbiology.
257-263.

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Undergraduate Research

  • 1. 1 Optimizing the Freshwater Denitrification Process of Heterotrophic Bacteria Using Iron Nanoparticles Katie Haske With Mercedes Dick, Ashley Hollinshead, and Carolyn Mazzuca CHE 480 Abstract Urgency to remove contaminants from the environment is becoming dire as we discover how harmful each is to life sustainability. Nitrate is a health-endangering carcinogen, and is therefore the focus of our experimentation. This research group tested the effectiveness of Pseudomonas aeruginosa, in conjunction with zero-valent iron nanoparticles, at reducing nitrate in water by converting it to ammonia and elemental hydrogen gas. The nanoparticles were created through the impregnation of iron salt solutions into environmentally-friendly milo seed carbon black. After these samples underwent Carbothermal reduction, each was combined with cultured P. aeruginosa and a 10mM nitrate solution and left to react for 46 hours. Samples were run in triplicates at 12°C, 25°C, and 35°C, and repeated at 35°C with initial nitrate solution concentrations of 0mM, 50mM, and 100mM NO3. The results yielded no significant change in nitrate or ammonia concentration between the various temperatures and initial NO3 concentrations.
  • 2. 2 Introduction Nitrate is a known carcinogen that is common to freshwater systems due to agricultural and industrial runoff (Shin & Cha 2008). Lives and environments worldwide, including the Emiquon Nature Reserve in Illinois, are being negatively impacted by this toxin as populations require more farmland and fresh water to sustain life. Though health problems have not been decisively proven, correlations have been found by the EPA resulting the nitrate concentration in drinking water required to be 10mg1-1 NO3 - -N or below (Shin & Cha 2008). Various means of reducing nitrate concentration in water have been used, such as ion exchange and reverse osmosis. However, each of these have posed economical complications due to operational costs and/or waste removal issues (Shin & Cha 2008). An alternative water treatment process to the previously mentioned cost-ineffective and waste-abundant choices is using agent zero-valent iron (ZVI). ZVI has been proven to be a powerful reducing agent of pollutants, but inevitable drawbacks exist. The equation below displays how the iron works: NO3 - + 4Fe(0) +10H+ → 4Fe2+ + NH4 + + 2H2O The ZVI reduces the nitrate into ammonia, which in high concentrations can also be dangerous to biological systems. Though the process requires acidic conditions and introduces excess ammonia into water systems, it is more cost-effective and green than existing processes, especially when the ZVI is produced from carbon black made from sustainable milo seed (Bapat, Manahan &Larsen 1999). Biological denitrification, executed with Pseudomonas aeruginosa, is an additional process option in reducing the nitrate concentration of freshwater systems. These bacteria are hardy and can survive under various, “hostile conditions” (Stoodley, Costerton & Stoodley
  • 3. 3 2004). With this choice, the nitrate is reduced to harmless elemental nitrogen, instead of ammonia which can accumulate and interrupt biological systems (Shin & Cha 2008). However, the unnatural addition of biological material will still impact and alter the nature of the area undergoing treatment. P. aerurinosa has been known to be cause dangerous infections in humans, so it must be handled with care and in careful concentrations (Hauser & Rello 2003). This process, like ion change and reverse osmosis, results in denitrification, as well as an increase in unwelcome materials into the water system—infectious bacteria and nanoparticles— requiring the system to undergo further treatment (Klaper). In a study by Shin and Cha, zero-valent iron works more effectively in a nanoparticle form. The size introduces numerous benefits in the process such as increased surface area and larger versatility in application location. The zero-valent iron nanoparticles (NZVI), in conjunction with nitrate reducing culture, are expected to work as displayed in the following reaction: 2NO3 - + 5H2 → N2 + 4H2O + 2OH- Rather than reducing the nitrate into ammonia, it can be reduced to innocuous nitrogen gas with the use of biotic reduction. The bacteria use the electrons from the NZVI to make this reaction occur (Park & Yoo 2009) . The path to this type of denitrification begins by creating sustainable BioChar. Carbon, in it’s activated, porous form, is an effective component in different processes of environment purification. It is a compelling component in decontaminating water, air, and other materials. Coal, wood, lignite, and agricultural wastes are only a few raw materials that can be used to create activated carbons (Bapat, Manahan &Larsen 1999). Because this material is so commonly used in decontamination processes, deactivated, used carbon itself becomes a
  • 4. 4 contaminant because of its abundance. From an economic and environmental position, reactivating spent carbon has become crucial in order to reduce problems associated with transportation of, disposal of, and cost of spent activated carbon (McGowin 1991). This experiment utilizes the ChemChar reverse-burn gasification of Milo seed (sorghum vulgare) to return deactivated carbon to its favorable, porous identity (McGowin, Kinner & Manahan 1991). Milo seed is a green, biological alternative used for activated carbon production, a component significantly less harmful to the environment than typically-used charcoal. The reactivation process consists of three thermal steps: 1. water and volatile adsorbates vaporization at 200°C, 2. additional volatile adsorbate vaporization and decomposition at 200°C-500°C, and 3. remaining adsorbate pyrolysis and activated char production at 500°C-700°C (McGowin 1991). The resulting carbon, if conducted successfully, will be once again useful in material purification due to its returned high-surface area, high-absorptive identity (Bapat, Manahan &Larsen 1999). This activated carbon is used to produce the NZVI. The method is simple: react soluble iron salts with carbon black, or thrice gasified milo Biochar, as described above. Two iron salt solutions, ferric nitrate and iron (III) citrate, were created, combined with the char, and placed through a Carbothermal reduction to produce the reactive and highly sorptive zero-valent iron nanoparticles (NZVI) that can remediate organic compounds that it comes into contact with (Hoch, et al. 2008). In conjunction with the selected bacteria, P. aeruginosa, nitrate concentration in water is expected to be reduced. The intention of this experiment determine the optimal conditions that bacteria denitrifies in conjunction with NZVI. Temperature conditions were varied to simulate real-world temperatures. The anaerobic process will be tested at 12°C, 25°C, and 35°C, representing
  • 5. 5 potential weather conditions. A second experiment was conducted at 35°C to observe how varying initial nitrate concentrations with 0mM, 50mM and 100mM solutions affects final nitrate reduction and ammonia production. Experimentation ChemChar Gasification Process Milo seed was baked in 9”x13” aluminum trays for at least 24 hours at 160°C. The bottoms of the trays were evenly covered in about a 1” thick layer of seeds, with all non-milo components removed. The baked tray of milo was removed, stirred, redistributed evenly along the bottom, and then returned into the oven at 220°C for an additional minimum of 24 hours. The twice-baked milo seed was then stored until ready to execute the ChemChar gasification process. To set up the ChemChar apparatus, a hollow iron tube was mounted in a vertical position. Depending on the amount of baked milo seed, this tube was occasionally substituted with a thinner Pyrex tube to ensure that the milo could reach the top of the tube when filled. 2-3 inches of fiberglass insulation was pushed into the bottom of the iron tube followed by a rubber stopper with a glass tube in the center (to eventually be attached to the compressed oxygen tank). Once this was set-up, baked milo seed was poured into the tube until it reached about 1 inch from the top. If there was not enough milo to fill the tube to the top, the thinner glass tube was used. It was best to pour the seed through a wide-necked funnel so that large chunks could be crushed with a spatula before entering the tube. The more uniformly sized the material, the more evenly it burned. As the seed was added, the column was packed by gently tapping it with a hand to compress it evenly. With rubber tubing, the glass tube in the stopper was attached to the compressed oxygen tank.
  • 6. 6 The compressed oxygen tank was then turned on enough to establish a gentle flow through the iron tube. When the oxygen was too high, milo seed was blown off the top; when the flow was too low, the milo could not sustain a flame front. When appropriate flow was established, the top layer was ignited with a small propane torch. Again, the oxygen was adjusted so that the flame front was a small, glowing simmer. The speed of the flame was tracked by spraying deionized water on the side of the iron tube. The position at which the water evaporated immediately revealed how far along in the iron tube the flame front was. Once it had reached the location of the fiberglass insulation, the oxygen was turned off, quenching the flame. This concluded the first-gasification of the milo seed. To gasify the milo seed a second time, the ChemChar apparatus was constructed identically to the first gasification, but instead the vertical tube was filled with the once-gasified milo seed; rather than immediately pouring the once-gasified milo into the iron tube, the milo was weighed, and 5% by weight of nanopure water was mixed into the seed. The water was poured on top of the milo and mixed in with a spatula carefully as to not to crush the milo. At this point, the seed was transferred into the iron tube and heated analogously to the first- gasification process. The third gasification was executed similarly, but with a 15% by weight addition of nanopure water. This thrice gasified BioChar was then stored until it was used in the impregnation process. Zero-Valent Iron Nanoparticle Synthesis Two iron solution were made: 2.6mM ferric nitrate solution and another with a 1.5mM iron (III) citrate solution. Each was then capped with Parafilm and inverted repeatedly until the salts were completely dissolved into the solution. Next, the BioChar was prepared.
  • 7. 7 The thrice-gasified BioChar was grinded with a mortar and pestle until the char was a fine powder. 6.25g of this ground BioChar was added to a 500 mL purged glass jar, as well as the ferric nitrate solution. In the same fashion, 5.12g of ground BioChar was added into a second purged 500 mL glass jar with the iron (III) citrate solution. Each of these jars were then placed into an automatic shaker at 200RPM for 24 hours. Once the time was up with the shaker, the solutions were put through separate vacuum filtration systems. To set up this apparatus, a rubber stopper was placed into the opening of a 250 mL side-arm flask, and then a Buchner funnel was placed inside that. An appropriately sized filter paper was put inside the funnel and secured down with a small amount of corresponding iron salt solution. The side arm of the flask was connected to the vacuum system with a rubber tube. The vacuum was turned on enough to create a gentle suction. Little by little, the iron solutions were poured into the vacuum filtration system. Rotating the jar while pouring had helped ensure that all of the impregnated BioChar was transferred into the funnel since it often adhered to the sides. This process was identical for both solutions. When the solid was dried, it was transferred into a storage container (after scraping as much off the filter paper as possible) after recording the weight for the yielded product. Carbothermal Reduction of Iron Salts Materials necessary for this process were a tube furnace, an asbestos tube, compressed argon tank, two rubber stoppers, a compressed gas regulator, a mineral oil bubbler, two ceramic boats, two glass jars with lids, and a spatula. Each of the dry iron salt solutions collected above were transferred into separate ceramic boats until the entire bottom of each respective boat was covered completely by an even layer of
  • 8. 8 salt. The boats were then slid into the center of the asbestos tube so that they were side by side. This tube containing the ceramic boats was then returned to the tube furnace, each side was plugged with a high-vacuum greased rubber stopper with a glass rod in the center. One stopper was connected to the compressed argon tank and the other was attached to the mineral oil bubbler, each with rubber tubing. At this point, the gas tank was turned on slowly—enough to create a gentle bubble in the bubbler. The gentle bubble, which was found by watching the bubble pace in the mineral oil bubbler, was ideal to prevent the waste of argon gas with equal effectiveness of removing oxygen and creating positive pressure inside the asbestos tube. The apparatus was run under these conditions for one hour, and then the heat was turned on to 800°C without ceasing the flow of argon gas. With temperature high, the apparatus was left to heat for six hours. After this time was complete, the heat was shut off and the furnace was allowed to cool for approximately one hour. The samples were then stored in glass jars that had been purged for future use. X-ray diffraction followed this process to test the success of the Carbothermal reduction in producing zero-valent iron nanoparticles. The reduced iron samples were ground with an agate mortar and pestle until each was a fine powder, so that it can be used in powder x-ray diffraction (XRD). A small aluminum XRD disc with about a 1” diameter was covered on one side with double stick tape. The edges of the tape were trimmed with a razor blade. A thin layer of iron powder was spread onto the tape. This disc was then placed into the diffractor at 2° per minute until 100°. With successful detection of the iron through this process, it would be evident that NZVI was present in the samples and the experimentation could proceed.
  • 9. 9 Culturing Bacteria Before the culturing the bacteria, LB broth was created for the bacteria to develop in. LB broth is a nutrient rich broth that encouraged high levels of growth; however, potential nitrate in the broth likely skewed the final results, so a nitrate free broth was necessary. To create the nitrate-free broth, the following components were dissolved in 1L of nanopure water: 1.   Dextrose: 0.860g 2.   HEPES acid: 14.227g 3.   HEPES sodium salt: 7.8189g 4.   KH2PO4: 0.2982g 5.   MnSO4*H2O: 0.1001g 6.   FeCl2*4H2O: 0.4505g 7.   CaCl*2H2O: 0.0250g 8.   CoCl2*2H2O: 0.1902g To this solution, 1mL of second 1L solution was added. The second 1L solution was composed of the following components dissolved into 1L of nanopure water: 1.   CuCl2*2H2O: 0.0302g 2.   H3BO3: 0.0309g 3.   ZnCl2: 0.0525g 4.   MgCl2*6H2O: 0.1000g 5.   NiCl2: 0.0239g 6.   MnNa2O4: 0.0371g Once this nitrate-free broth was complete, 0.500M HCl was added until the pH was brought to 7.
  • 10. 10 5mL of each broth was put into several 18mm sterile glass test tubes. Bacteria was inoculated into six tubes of this broth once a week to ensure the bacteria is healthy and active. To accomplish this, the opening of a test tube and a small metal loop first were flamed in a Bunson burner for sterilization. The loop was then dipped into a supplied sample of bacteria (inoculated in LB broth) and transferred into a test tube with nitrate-free broth. The test tubes were promptly recapped and the metal loop sterilized in the Bunsen burner flame. This process was repeated every week with six test tubes of new nitrate-free broth. Once transferred, the test tubes were placed into a 35°C incubator for 24 hours and then into a refrigerator until needed, as recommended by previous literature (Williams, Rowe, Romero & Eagon 1978). Creating Solutions for Experiment 1 and Experiment 2 For the trials conducted at varying temperatures, experiment 1, 10mM nitrate solution was created from solid KNO3 and nanopure water. For experiment 2, solutions of 0mM, 50mM, and 100mM were created from KNO3 and nanopure water. Anaerobic Biotic Nitrate Reduction Process For experiment 1, there were 3 trials and a control—one Wheaton jar per sample. To each of these jars, 6mL of the nitrate solution was added, 54mL of nitrate-free broth, which were measured in appropriately-sized graduated cylinders, and 0.30g of iron nanoparticles. Because of the danger of the bacteria, the following steps were executed in a Laminar flow hood. Five test tubes of bacteria-filled nitrate-free broth were poured and mixed into a 250 mL Erlenmeyer flask until the solution was uniform. With a 5 mL plastic sterile volumetric
  • 11. 11 pipet, 5 mL of bacteria solution was added into the Wheaton jars containing samples 1, 2, and 3. An additional 3mL of of bacteria solution was pipetted into a scintillation vial to test for bacteria presences with the DAPI process later. Time zero-samples were taken with autoclaved 12-inch long needles attached to sterile syringes to draw up four 20 mL samples, one from each Wheaton jar, and dispense each into individual whirl-packs. These were then stored in the freezer for future analysis. Next was the process for collecting time-final samples. Small syringes that were stuffed with cotton (to vent the solutions without contaminating the air with bacteria) were pierced through the top of each Wheaton jar. The previously used 12-inch needles were made sure to be submerged into the solutions, and then connected to tubing that was connected to a nitrogen gas tank. The jars were purged for 15 minutes with nitrogen gas. When this time was complete, both the 12-inch needles and the small syringes were removed, the gas was shut off, and the holes in the lids were sealed with grease. These jars were then put into a shaker for 46 more hours at 200RPM, and then the solutions were transferred to desterilized falcon tubes for later analysis. This process was repeated for each temperature trial (12, 25, and 35°C) in experiment 1, as well as for all the trials in experiment 2; however, 11 jars underwent this process in experiment 2 rather than four. Three concentrations were tested: 0mM NO3, 50mM NO3, and 100mM NO3. Each concentration consisted of 3 replicates (composed of nitrate solution, nanoparticles, and bacteria) and 1 negative control (composed of solution and nanoparticles). Additionally, 3 positive controls (composed of nanopure water, nanoparticles, and bacteria) were tested.
  • 12. 12 Phenate Method for Ammonia Concentration Determination To determine how much ammonia was produced over the course of this experiment, a series of solutions were made to be run through a spectrophotometer to test for absorbance (Barletti, Paver, & Dungey 2007). The following solutions were made: 1.   Phenol/Ethanol: Dilute 5.55 mL phenol in a 50 mL volumetric flask with 95% ethanol 2.   Sodium nitroprusside solution 0.5%: Dissolve 0.5 g of sodium nitroprusside in 100 mL of nanopure water 3.   Alkaline citrate: Dissolve 200 g of trisodium citrate and 10 g of sodium hydroxide in 1 L of nanopure water in a 1L volumetric flask. 4.   Sodium hypochlorite 5-6%: Use commercial bleach 5.   Oxidizing solution: Mix 100 mL of alkaline citrate solution with 25 mL of sodium hypochlorite solution 6.   Stock ammonia solution: Dissolve 3.819 g of anhydrous ammonium chloride (dried at 105° C) in 1 L of nanopure water Twenty 20 mL scintillation vials were then labeled appropriately. The following 7 standards were made using an ammonia working stock solution and put into scintillation vials. Working stock solution: 0.10 mL of stock ammonia solution was added to 100 mL volumetric flask and bring to volume with nanopure water Blank: 15 mL of nanopure water 1.   15 mL of working stock solution 2.   Add 7.5 mL of working stock solution to 7.5 mL of nanopure water 3.   Add 3 mL of working stock solution to 12 mL of nanopure water 4.   Add 1.5 mL of working stock solution to 13.5 mL of nanopure water
  • 13. 13 5.   Add 750µL of working stock solution to 14.25 mL of nanopure water 6.   Add 375µL of working stock solution to 14.625 mL of nanopure water 7.   Add 150µL of working stock solution to 14.85 mL of nanopure water In addition to the standards, solutions with the samples were made. With a 15 mL disposable serological pipette, 15 mL of each sample at To and Tf was pipetted into respective scintillation vials. 600µL of sodium nitroprusside solution was pipetted into each vial (both the vials with the standards and the vials with the samples) using an automatic micropipettor. 1.5 mL of the oxidizing solution was then added to all the scintillation vials, as well. Once all solutions and standards were complete, the vials were capped, inverted three times, and covered completely with aluminum foil to protect the solutions from light. The covered vials were left to sit for a minimum of one hour. When the setting time was complete, the absorbance’s of all vials were measured using a Spec 20 Geneysis spectrophotometer at 640nm. This process was repeated for the samples collected from experiment 2. Ion Chromatography Method for Nitrate Concentration Determination Ion Chromatography was used to determine the nitrate concentration of the time initial and time final samples (Dungey 2013). Standard solutions were created using ion chromatography standard 6. In IC Vials, standards were diluted with 5, 50, 100, 500, and 2000µL up to 5mL with nanopure water in IC vials. 50µL of each sample, time-initial and time- final, collected was diluted up to 5mL with nanopure water into IC vials. Each vial was placed into the chromatograph for analysis. This process was repeated for the samples collected from experiment 2.
  • 14. 14 DAPI Protocol for Bacteria Quantification The DAPI protocol is a procedure used to quantify the bacteria in samples (Dungey 2016). The 3mL bacteria sample preserved from the Anaerobic Biotic Nitrate Reduction Process was used in this process. To set up this procedure, a 25mm diameter fritted glass filter was secured onto the neck of a 250mL side arm flask with plastic clamps. A 0.22𝜇m pore backing filter was placed on the glass filter, followed by a 0.22𝜇m pore, black membrane filter, and finally a second fritted glass filter. With rubber tubing, this system was attached to a vacuum at 5PSI. The following materials were added to the funnel in numbered order: 1.   1mL nanopure water 2.   Sample a.   LB broth samples were diluted to 10𝜇M with saline b.   Nitrate-free broth sample was diluted to 100𝜇M 3.   200𝜇L DAPI With the vacuum still on and these components added to the filter, the system was covered with aluminum to block out light and left to react for 5 minutes. Once this was complete, the set-up was disassembled, and the membrane filter was placed onto a microscope slide overtop a drop of FF immersion oil. A second drop of oil was placed on top of the filter, followed by the slide cover. With a Kimwipe, the cover was gently pressed down, flattening the layers between the glass. The slide was then viewed under a microscope.
  • 15. 15 Results Figure 1. Powder X-Ray Diffraction of iron nitrate sample. Sample was at 2 degrees per minute at 2.29 angstroms. Peak at 69°C identifies successful production of iron nitrate nanoparticles following nanoparticle impregnation of Biochar. Temperature Sample Absolute Difference (mg/L) Percent Difference Average Percent Difference +/- 1 SD (without control) 12°C Control -0.8887 -45.3 -14 +/- 101 -0.3716 -28.9 2 -0.0437 -4.1 3 -0.1002 -8.6 25°C Control +0.3129 +36.1 -22 +/- 141 -0.8359 -41.1 2 -0.1801 -13.7 3 -0.1299 -10.0 35°C Control -0.0113 -1.1 -4 +/- 51 -0.0238 -2.5 2 +0.0029 +0.3 3 -0.1033 -10.2 Table 1. Nitrate Change Results The nitrate concentration change results, from time initial (T0) to time final (TF) collected through ion chromatography. 5 samples of varying concentrations of IC standard 6, and of each control and sample diluted from 50µL to 5µL were collected analyzed with Dionex ICS 2000.
  • 16. 16 Absolute difference in concentration was calculated by subtracting T0 raw value from T0 raw value. Percent difference was calculated by subtracting TF from T0, dividing the difference by T0, and multiplying the value by 100%. The standard deviation was calculated excluding the control. Nitrate 10 20 30 Temperature (°C) Figure 2. Percent Change in Nitrate Concentration The blue circles represent the samples, the pink triangles represent the control (samples lacking Pseudomonas aeruginosa), the error bars are ± one standard deviation, and the gray bars are the average percent change in concentration all with respect to temperature groups. -­‐50 -­‐40 -­‐30 -­‐20 -­‐10 0 10 20 30 40 50 0 10 20 30 40 PercentChange(%)
  • 17. 17 Temperature Sample Absolute Difference in Concentration (mg/L) Percent Difference in Concentration Average Percent Difference +/- 1 SD (without control) 12°C Control +0.0008 -2.89 23 +/- 101 -0.0100 +20.5 2 -0.0179 +33.0 3 -0.0163 +40.3 25°C Control -0.0109 +41.1 12 +/- 851 -0.0161 +100. 2 +0.0133 -53.9 3 +0.0038 -38.1 37°C Control +0.0009 -6.85 -54 +/- 341 -0.0121 +36.3 2 -0.0215 +53.7 3 -0.0181 +73.4 Table 2. Ammonia Change Results The ammonia absorbance change results, from time initial (T0) to time final (TF) collected through the phenate method and run through a spectrophotometer. Absolute difference in absorbance was calculated by subtracting T0 raw value from TF raw value. Percent difference was calculated by subtracting TF from T0, dividing the difference by T0, and multiplying the value by 100%. The standard deviation was calculated excluding the control.
  • 18. 18 Ammonia Figure 3. Percent Change in Ammonia Concentration The blue circles represent the trials, the pink triangles represent the control, the error bars are ± one standard deviation, and the gray bars are the average percent change in concentration all with respect to temperature groups. -­‐175 -­‐150 -­‐125 -­‐100 -­‐75 -­‐50 -­‐25 0 25 50 75 0 5 10 15 20 25 30 35 40 PercentChange(%) Temperature (oC)
  • 19. 19 Nitrate Concentration of Initial Solution (mg/L) Sample Absolute Change in Concentration (mg/L) Percent Change in Concentration (mg/L) Average Percent Difference +/- 1 SD Positive Control 1 0.0033 -132 -219 +/- 980 Positive Control 2 0.0056 -200 Positive Control 3 0.0052 -325 Negative Control -0.0804 1.21 -3 +/- 5 50 1 0.1478 -2.52 2 0.4287 -7.84 3 -0.0857 1.54 Negative Control -0.2701 2.14 1 +/- 8 100 1 -0.5638 4.70 2 0.9102 -8.00 3 -0.7826 6.55 Table 3. Trial 2 Nitrate Change Results Trial 2 was run at 35°C to maximize resulting affect of initial nitrate concentration on final nitrate concentration. Positive control consisted of nanopure water, NZVI, and bacteria, negative control consisted of solution and NZVI, and the replicated consisted of solution, NZVI, and bacteria.
  • 20. 20 Figure 4. Trial 2 Percent Change in Nitrate Concentration The blue dots represent the replicates per concentration trial, the pink dots represent the negative control per respective trial, the grey bars represent the average, and the pink triangles represent the control. Nitrate Concentration of Initial Solution (mg/L) Sample Absolute Change in Concentration (mg/L) Percent Change in Concentration (mg/L) Average Percent Difference +/- 1 SD 0 Positive Control 1 0 0 9 +/- 9 Positive Control 2 -0.0033 +10.4 Positive Control 3 -0.0065 +17.2 50 Negative Control -0.0131 +31.6 22 +/- 71 -0.0065 +15.5 2 -0.0115 +23.9 3 -0.0147 +28.3 100 Negative Control -0.0106 +23.4 35 +/- 201 -0.0131 +27.3 2 -0.0090 +19.8 3 -0.0303 +58.2 Table 4. Trial 2 Ammonia Change Results -­‐350 -­‐300 -­‐250 -­‐200 -­‐150 -­‐100 -­‐50 0 50 -­‐20 0 20 40 60 80 100 120 Percent  Change  (%) Initial  Nitrate  Concentration  (mg/L) Percent  Change  in  Nitrate
  • 21. 21 Trial 2 was run at 35°C to maximize resulting affect of initial nitrate concentration on final nitrate concentration. Positive control consisted of nanopure water, NZVI, and bacteria, negative control consisted of solution and NZVI, and the replicated consisted of solution, NZVI, and bacteria. Figure 5. Trial 2 Percent Change in Ammonia The blue dots represent the replicates per concentration trial, the pink dots represent the negative control per respective trial, the grey bars represent the average, and the pink triangles represent the control. -­‐10 0 10 20 30 40 50 60 70 -­‐20 0 20 40 60 80 100 120 Percent  Change  (%) Initial  Nitrate  Concentration  (mg/L) Percent  Change  in  Ammonia
  • 22. 22 Figure 6. Bacteria Quantification Images of Pseudomonas aeruginosa taken through optical lens of microscope available through the DAPI protocol. The left image displays illuminated bacteria grown in LB broth, and the left image displays illuminated bacteria grown in low-nutrient nitrate-free broth. Discussion Figure 1 displays the powder x-ray diffraction results of the iron nitrate samples. The peak just below 70°C indicates the successful production of zero-valent iron nanoparticles. With the existence of NZVI in the samples, it was predicted that nitrate will be reduced, with or without additional biological denitrification. The supplemental biological component, P. aeruginosa is displayed in Figure 4. Through the DAPI process, existing bacteria in the samples were clearly illuminated under a microscope. The bacteria cultured in the LB broth was thriving as opposed the that cultured into the nitrate-free broth—the broth used throughout experimentation. This broth was made of various components and only a small amount dextrose that the bacteria could survive from; it was likely that the bacteria was starved. Additionally to the DAPI process to determine existence of bacteria, Anna Ball, biology student at the University of Illinois at Springfield, did gram stains of nitrate-free broth cultures confirming the lack of
  • 23. 23 active or inactive bacteria in the solutions. The samples relied solely on the NZVI for nitrate reduction and ammonia production. The percent change in nitrate concentration (see Table 1 and Figure 2) of the triplicate samples and the respective controls behaved oppositely. At 12°C, the control reduced the nitrate significantly more than each of the samples, opposite that of the samples ran at 25°C. The control was within the narrow sample array of 35°C, clustered around 0% change. The overall error range for the change in nitrate concentration was consistent with zero percent change. This suggests that temperature has no effect on the reduction of nitrate in 100mM nitrate solutions over 46 hours. This could be due possibly to the lack of bacteria, time that samples were allowed to react, or the concentration of the nitrate solutions that the nanoparticles and bacteria were working in. Table 2 and Figure 3 display the percent change in the ammonia concentration over the course of the experiment. Though the control of each temperature trial was above the average of the corresponding sample averages, opposite of what was expected, the range and behavior of the samples made this trend unreliable to conclude from (Shin & Cha 2008). The controls of 12°C and 35°C were significantly larger and of opposite sign of the corresponding triplicate samples. Though the control of the 25°C trial is close to average of the samples, the span of percent change ranges over 150% with two of the samples falling beyond the error bars, double that of both the other trials. 4 out of the 9 samples and 2 of the 3 controls were placed beyond the error bars of the respective temperature. This was predicted to be the results of inappropriate initial nitrate concentration selection that the nanoparticles and bacteria were reacting in. A final trial experimental section (see Table 3 and Figure 4) with varying initial nitrate solution concentrations was conducted to quantify how much the change in nitrate and ammonia
  • 24. 24 was affected by this concentration. Three concentrations were tested: 0mM NO3, 50mM No3, and 100mM NO3. Each concentration consisted of 3 replicates (nitrate solution, nanoparticles, and bacteria) and 1 negative control (solution and nanoparticles). Additionally, 3 positive controls (nanopure water, nanoparticles, and bacteria) were tested. The nitrate percent change of the positive controls varied greatly from the other concentrations. Even though this solution had a nitrate concentration of 0mM, the average percent difference of nitrate was -219%, compared to the nitrate solutions having average percent differences of -2.9% and 1.1%. Additionally, the nitrate solutions behaved oppositely: the 50mM solution had a decrease in nitrate concentration and the 100mM solution had an increase in nitrate solution. This trial suggests that nitrate concentration has no effect on nitrate reduction. The percent change in the ammonia for each concentration mostly clustered closely; however, there is no significant difference across trials. The positive controls had a smaller increase in ammonia that the other solutions, as expected. However, the negative controls without bacteria behaved so similarly to the replicates with bacteria that the impact of the bacteria appeared to be negligible, further confirming the lack of bacteria in the solutions as displayed in Figure 4. Initial nitrate concentration has no effect on how much ammonia is produced or reduced. Conclusion The purpose of this experiment was to define optimal conditions that zero-valent iron nanoparticles in conjunction with the bacteria Pseudomonas aeruginosa could denitrify water systems more efficiently with the intention of eventually applying it to the Emiquon Nature Preserve in Illinois. NZVI was successfully created through the various processes described, but
  • 25. 25 particles were not as effective as expected. The affect of bacteria on this system is inconclusive since did not survive through the trials. Neither temperature nor initial nitrate concentration were proven to affect how the concentration of the nitrate or the ammonia changed after anaerobic exposure to NZVI and bacteria. However, future research will benefit from this project. A different type of bacteria, such as an active sludge, is recommended. An already active denitrifying biological material is likely to reduced nitrate concentration in water systems with the addition of NZVI. This will eliminate many of the suspected errors that existed throughout this project and could be the solution to denitrifying contaminated freshwater systems.
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