dephosphorylation via metal oxides with a focus on cobalt …
TRANSCRIPT
DEPHOSPHORYLATION VIA
METAL OXIDES WITH A FOCUS ON
COBALT OXIDE
By
Wilhelm Liano
A thesis submitted to Johns Hopkins University in conformity with the
requirements for the degree of Master of Science
Baltimore, Maryland
May 2018
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Abstract: Global phosphorus usage is steadily increasing as the human population continuously
grows; consequentially, the amount of phosphorus wasted proportionally increases. To
counteract this problem, a way to capture and recycle as much of the lost phosphorus is vital for
the longevity of this important element.
Here I report on methods to synthesize different metal oxide nanoparticles with a focus on cobalt
oxide to promote successful dephosphorylation. Various characterization techniques are done to
identify each metal oxide and its catalytic properties. The dephosphorylation reaction was further
studied across multiple cobalt oxide morphologies at various temperatures to provide kinetic
results unique to each morphology along with accompanying factors affecting the reaction
efficacy. My results show that the most efficient catalyst boasts about a 92% conversion rate on
phosphorus extraction. Additionally, a recyclability study shows the reusability of the cobalt
oxide nanoparticles which remain with conversion rate of approximate 97% of the previous
yield.
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Acknowledgments
To anybody reading this, I am glad my work in preserving phosphorus reserves is of interest to
you. This work could not have been possible without the support of family, friends, colleagues,
and professors and staff in the Johns Hopkins Chemical and Biomolecular Engineering
department. A huge thank you goes to the friends and lab-mates in Dr. Chao Wang’s lab group,
as without everyone’s guidance, support, criticism, and company, my interest in research would
not have flourished. A special thanks to Michael Manto, Pengfei Xie and the rest of our
heterogeneous catalysis subgroup, as without their help, none of what I have accomplished
would have been possible. I want to thank Dr. Chao Wang for mentoring me and allowing me to
work in his space to explore catalysis and nanotechnology since the beginning of 2015.
Finally, I want to thank Dr. Michael McCaffery and the JHU IIC, as they have been excellent in
helping me finalize the characterizations with microscopy throughout my years here.
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Table of Content:
I. An Introduction to Phosphorus
A) Phosphorus Status Quo
B) Environmental Impact
C) Reincorporating Lost Phosphates
II. Background to Dephosphorylation
A) Mechanism
B) Model Reactant
III. Various Metal Oxide
A) Choosing Metal Oxides
a. What makes Metal Oxides Good?
b. Oxygen Vacancies
B) Metal Oxides
i. Synthesis and Characterization
ii. Results
IV. Cobalt Oxide
A) Why Cobalt Oxide?
B) Cobalt Oxide Morphology Study
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a. Nanospheres
b. Nanocubes
c. Nanorods
C) Results
D) Kinetics
E) Recyclability
V. Conclusion/Future Works
a. Results Summary
b. Alternative Metal Oxides
c. Environmental Significance
d. Improvements
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List of Figures:
Figure 1: Estimated trend of phosphorus production, use, and waste over the years. Ingraph:
Estimated trend of phosphorus rock mining production. ............................................................... 2
Figure 2: A massive algal bloom of cyanobacteria in Lake Erie. Over 700 square miles of algae
covered Lake Erie, turning the lake mostly bright green. Picture taken September 26, 2017 ....... 3
Figure 3: Scheme of the dephosphorylation mechanism of para-nitrophenyl phosphate into p-
nitrophenol and a phosphate ion, where the orange bond is cleaved. Further pH treatment
degrades p-nitrophenol into p-nitrophenolate in a basic solution of at least pH 7.5 ..................... 6
Figure 4: A) supernatant taken from the dephosphorylation of p-NPP into p-NP, demonstrating a
stronger yellow color the higher the concentration of p-NP. B) Supernatant treated with
molybdenum blue assay, demonstrating a darker blue color the more phosphate present in the
solution ........................................................................................................................................... 7
Table 1: A trend showing the P-O ester bond energy (EbP-O) normally, and the activation energy
of P-O scission (EA) on ceria octahedra. ....................................................................................... 8
Figure 5: Trend of average vacancy formation energy for metal oxides which should represent
the best metal oxides to catalyze dephosphorylation. .................................................................. 10
Figure 6: Synthesis scheme of the various metal oxides ............................................................ 12
Figure 7: Transmission electron microscopy of a) Vanadium oxide (V2O5), b) Cobalt oxide
(Co3O4), c) Samarium oxide (Sm2O3), d) and Lanthanum oxide (La2O3) ................................... 13
Figure 8: X-ray diffraction of metal oxides as confirmation of the composition of a) Samarium
oxide (Sm2O3) b) Lanthanum Oxide (La2O3) c) Cobalt oxide (Co3O4) d) Vanadium oxide (V2O5)
...................................................................................................................................................... 15
Figure 9: A) yield of p-NP at 25oC of metal oxides tested in addition to cerium oxide B) and
yield of phosphate at 25oC of metal oxides tested in addition to cerium oxide. .......................... 16
Figure 10: Poisoning of phosphate onto the surface of metal oxides from initial reaction. ....... 17
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Figure 11: A) Transmission electron microscopy of ~20nm cobalt oxide nanospheres. B) X-
ray diffraction of the cobalt oxide nanospheres ........................................................................... 20
Figure 12: A) Transmission electron microscopy of 30-50nm cobalt oxide nanocubes. B) X-
ray diffraction of the cobalt oxide nanospheres ........................................................................... 21
Figure 13: A) Transmission electron microscopy of 200nm wide cobalt oxide nanorods. B) X-
ray diffraction of the cobalt oxide nanospheres ........................................................................... 23
Figure 14: Experimental yield of various morphologies of cobalt oxide for a) p-NP b) phosphate
...................................................................................................................................................... 25
Figure 15: The amount of phosphate that poisoned the catalyst, found by the difference between
the yields of p-NP and phosphate. ............................................................................................... 26
Figure 16: Plot of the reaction rate of dephosphorylation using cobalt oxide nanospheres at
various temperatures as a demonstration. .................................................................................... 28
Figure 17: Arrhenius plot showing the variance of rate constant k dependent on temperature . 29
Figure 18: Activation energies derived for various morphologies of cobalt oxide. ................... 30
Figure 19: a) Recyclability of catalysts shown by the amount of phosphate yield after each run,
b) and a graph displaying the average percentage difference in phosphate yields after all the runs.
...................................................................................................................................................... 32
Figure 20: Modelled wastewater treatment plant with the intent of incorporating cobalt oxide
nanospheres combined with Cu-ZSM5 to dephosphorylate, capture, and release excess
phosphates in the sludge .............................................................................................................. 35
1
I. An Introduction to Phosphorus
A) Phosphorus status quo
Phosphorus is a vital and nonrenewable resource necessary to provide food for the global
population. As the population reaches 7.6 billion people and rising, the amount of food
needed to produce correspondingly needs to match and increase. However, the production of
phosphorus is limited to mining and introducing fresh phosphorus into the cycle while over
10% the amount of phosphate used was lost due to waste in food, runoff, and released
wastewater discharge.1 Consequentially, over 80% of phosphorus resources are derived from
phosphorus rock mining. As phosphorus is a nonrenewable resource, these rocks will
eventually reach depletion, where experts estimate the peak mining of these rocks will be
around the year 2040 [Figure 1].2 Further analysis provides that total depletion of the
phosphorus rocks, accommodating for the yearly increase in population, will occur around
the year 2100. Once this point is reached, the amount of food able to be grown will decline
and will no longer be able to support the population growth.
2
Figure 1: Estimated trend of phosphorus production, use, and waste over the years. Ingraph:
Estimated trend of phosphorus rock mining production
B) Environmental Impact
All the wasted phosphorus ends up somewhere harmful. One widespread environmental problem
concerning America is the nutrient pollution. This is caused by an excess of elements such as
nitrogen and phosphorus in both the air and in the water. Both are vital components for
prosperity in crops and plants. However, in excess, polluted air and water promote the rapid
growth of algae; in large bursts, these are called algal blooms. These are extremely hazardous to
the environment, notably any large body of water. The algal bloom heavily reduces the oxygen
concentration in water, leading to illness and even death for the aquatic wildlife. While this alone
is concerning, certain algal blooms are even toxic to humans. Cyanobacteria, also known as blue-
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green algae, produce toxins that are harmful to people. In 2014, a large concentration of
cyanobacteria bloomed and poisoned a water treatment plant in Toledo, Ohio. Drinking water in
Toledo was shut down for 3 days after the incident. While water treatment plants are a rarer case,
Lake Erie itself is susceptible to these toxic algal blooms, and they are becoming more common
as different farming practices are allowing the chances to spike. 3
Figure 2: A massive algal bloom of cyanobacteria in Lake Erie. Over 700 square miles of algae
covered Lake Erie, turning the lake mostly bright green. Picture taken September 26, 2017. 4
Nearby agriculture, most notably the corn belt, heavily influences the wasted phosphorus flowing
into nearby lakes such as Lake Erie. Metson et al. states that if 30% of the annual wasted
phosphate in the US is recycled, this would provide enough phosphate to supply the corn
industry for a year. Between vast amounts of waste in manure and human food, as well as the
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subsequent runoff from farming practices, these are the proposed places to focus phosphorus
recycling efforts. 5
C) Reincorporating Lost Phosphates
Despite knowing that phosphorus is a finite resource that is going to be depleted relatively soon,
few efforts have been made to preserving and recycling the phosphorus in use. The primary
challenge ahead lies in discovering and developing an efficient method to recycle any
phosphorus, as eventually all the rocks will be mined away. This means that all the waste from
the wastewater discharge or from crop runoff will need to find its way back into the phosphorus
cycle. Efforts need to be made to take the wastewater and runoff and isolate all useful elements
to filter them from the true waste. Phosphorus, nitrogen, even protein and carbohydrates exist in
current algal waste.6 Many labs grow and culture algae for research purposes regarding fuels,
derived from the carbohydrates and oils. Plenty of the nucleic acids, proteins, and other key
components of the algae are found to be “wasted,” where these components aren’t used for much
else and effectively tossed. As a result, a method needs to be developed where all the wasted
elements can be captured and introduced into the cycle. One method proposed is to
dephosphorylate the waste for capturing and reusing phosphates to extend the longevity of the
phosphorus resources described prior.
II) Background to Dephosphorylation
A) Mechanism
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Dephosphorylation of molecules is removing a phosphate group (PO4-3) group through
hydrolysis, a method of cleaving chemical bonds using water. Specifically, the enzyme
phosphatase is responsible for cleaving a phosphoric acid compound into a phosphate ion and an
alcohol. One of the most well-known examples is the conversion of adenosine triphosphate
(ATP) to adenosine diphosphate (ADP). As the name suggests, the process cleaves off one of the
phosphates, where the breaking of the bond creates energy along with a phosphate ion and is
then used as an energy source for a cell.
B) Model Reactant
In terms of compounds that are susceptible to dephosphorylation, ATP is a strong candidate.
However, a simpler and more straight-forward compound has been chosen instead. Para-
nitrophenyl phosphate (p-NPP) can be considered the “model” reactant due to its characteristics
before and after the dephosphorylation process. Shown in Figure 3 is a scheme of p-NPP
dephosphorylating into the phosphate ion and para-nitrophenol (p-NP), where in a basic solution
p-NP turns into p-nitrophenolate. To convert the p-NP into p-nitrophenolate, a minor pH
adjustment must be made. This is done by adding 30 µL of a 1% NaOH solution into 1mL of
supernatant. One reason that makes p-NPP such a great reactant to start with is that there exists a
negligible amount of side reactions that can occur. However, more notably, the initial solution of
p-NPP in an aqueous solution is a clear color. When it dephosphorylates, p-NP and phosphates
are formed, and the existing para-nitrophenolate turns the aqueous solution a yellow color, if the
solution has a pH of at least 7.5.7 This visual indicator is the simplest way to tell if any catalyst
worked. Furthermore, the dephosphorylated compounds are all in a theoretical 1:1 ratio, where if
p-NPP undergoes a reaction, there should be an equimolar amount of phosphate and p-NP
present to that of p-NPP dephosphorylated. Any less phosphate amount indicates a potential
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poisoning of the catalyst with phosphates. A demonstration of the visual reaction occurs over
time the stronger more p-NPP dephosphorylates [Figure 3]. To analyze each sample for
concentration of p-NPP, p-NP, and phosphate, a UV-Vis spectrophotometer is needed. Taking
the measurements of the supernatants at wavelengths of 310nm and 400nm measure p-NPP and
p-NP concentrations, respectively.8 A molybdenum blue assay [made by mixing 5mL of 4.0wt%
ammonium molybdate, 17mL of 5.0N sulfuric acid, and 10mL of 0.1M L-ascorbic acid] helps
measure the amount of phosphate in the supernatant. An addition of 0.2 µL molybdenum blue is
added to 1mL of supernatant to measure the amount of phosphate in the solution, and then goes
through the UV-Vis spectrophotometer measuring at wavelength of 890 nm.9 The stronger the
concentration of phosphate, the darker blue the solution will turn.
7
Figure 3: Scheme of the dephosphorylation mechanism of para-nitrophenyl phosphate into p-
nitrophenol and a phosphate ion, where the orange bond is cleaved. Further pH treatment
degrades p-nitrophenol into p-nitrophenolate in a basic solution of at least pH 7.5.
Figure 4: A) supernatants taken from the dephosphorylation of p-NPP into p-NP, demonstrating
a stronger yellow color the higher the concentration of p-NP. B) Supernatant treated with
molybdenum blue assay, demonstrating a darker blue color the more phosphate present in the
solution.
A) Choosing Metal Oxides
There are many examples of metals being used as an artificial enzyme. To fully promote a
dephosphorylation reaction, the catalyst of choice must also replicate an enzyme activity. Many
research papers go into detail on how many metal oxides can be used as some form of artificial
enzyme, where lanthanum can serve to cleave RNA sequences, or where samarium oxide serves
to promote methane coupling. [10,11] As a result, metal oxides are a strong contender for
mimicking enzymatic activity. In fact, there exists a reason why certain metal oxides may
perform the coveted dephosphorylation reaction, with further explanations on why specific metal
oxides outperform others.
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i) What makes metal oxides good?
A look from our paper on cerium oxide shows promising results that one of the main reasons that
cerium oxide is able artificially mimic a phosphatase is due to the oxygen vacancies present on
its surface. This theory is further supported by Zhao and Xu, where they suggests that cerium
oxide readily weakens the P-O ester bond enough so that the “P-O ester bond scission is
kinetically insignificant compared to phosphate hydration and desorption.”12 This is
demonstrated by analyzing the energy required to cause a P-O scission in many trials containing
different species and comparing the P-O bond energy to the activation energy for the bond
scission on cerium oxide.
Table 1: A trend showing the P-O ester bond energy (EbP-O) normally, and the activation energy
of P-O scission (EA) on ceria octahedra.12
ii) Oxygen vacancies
Something must be happening to the phosphate that promotes the P-O bond weakening. Zhao
and Xu further explain a potential mechanism to what reduces the activation energy to cleave the
P-O bond and support our theory that one key factor is the oxygen vacancies present on the metal
oxide catalyst. Density functional theory (DFT) calculations have concluded that it is highly
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likely that the orientation of the molecules containing the phosphate (p-NPP as an example) is
adjusted onto these vacancies in such a way that the P-O bond lengthens as the phosphate’s
negatively charged oxygen binds to the vacancy.
Furthermore, Ganduglia-Priovano et al. shows that the higher the formation energy for the
oxygen defects, the more stable these oxygen defects turn out to be.13 “The defect formation
energies in the TM oxides follow the trend: V2O5 < TiO2 (rutile) < TiO2 (anatase) < t-ZrO2 .”
They continue by stating that stability of specifically ceria is as follows regarding the surfaces:
(111) > (110) > (100), where the average energy of formation goes as follows: 3.61 eV (111),
2.94 eV (110), and 2.27 eV (100). Similar trends can be found in ZrO2. The (101) surface has
similar stability for two different methods, yielding an oxygen vacancy formation energy of 5.7
eV and 5.48 eV; due to such a relatively small energy difference, no conclusion can be drawn on
stability other than the known trend, as the defect stability was too similar. However, the stability
of the (001) surface is stated to be more stable, with a formation energy of 6.4 eV.
Understanding that the higher the oxygen vacancy formation energy, the more stable the surface
accompanied with knowing that the dephosphorylation process is correlated to the strength of the
oxygen defect stability, we can create a trend on which metal oxide should perform best in
dephosphorylation of our model reactant.
Metal oxides
When searching for a new catalyst, we searched for metal oxides with oxygen defects that were
similar to cerium oxide, as it would be a strong indicator being able to catalyze the
dephosphorylation reaction.
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Data from multiple sources have been compiled to show the formation energy of oxygen
vacancies. Here, we give a few metal oxides to consider. As Ganduglia-Pirovano has stated,
cerium oxide, vanadium oxide, titanium oxide, and zirconium oxide all have potential due to
their oxygen vacancy stability strengths. Furthermore, lanthanum oxide and samarium oxide
were chosen as data exists for their oxygen vacancy formation energy [Figure 4]. 14, 15 Further
research showed that samarium oxide and lanthanum oxide both have been used as an artificial
enzyme. 17, 18.
Figure 5: Trend of average vacancy formation energy for metal oxides which should represent
the best metal oxides to catalyze dephosphorylation. 14, 15, 16
To test multiple metal oxides, it is simplest to test commercial catalysts for the six metal oxides
mentioned above. Of these six, only four had worked; Zirconium oxide and titanium oxide had
zero results, as the p-NP and phosphate has no results when testing the corresponding
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wavelengths with the UV-Vis. Additionally, we tested iron oxide to see if it would work, as it is
a cheap metal oxide and it would be fantastic if it did work. Similar results showed no detection
of p-NP nor phosphate in the solution. Consequently, we had four metal oxides to work with:
Vanadium oxide, cobalt oxide, samarium oxide, and lanthanum oxide. Initial test results showed
that cobalt oxide and vanadium oxide had a relatively strong ability to dephosphorylate p-NPP,
with a p-NP conversion of 40% and 29% respectively. Samarium oxide and Lanthanum oxide
showed very little reaction, but a reaction nonetheless, with a meager 5% and 8% respectively.
i) Synthesis and Characterizations
However, there is no way of knowing exactly how effective the commercially synthesized
catalyst could be, so a method was developed to attempt to synthesize the catalysts needed to
test. As noted previously, titanium oxide, zirconium oxide, and iron oxide were inactive
regarding dephosphorylating our model reactant p-NPP, so these catalysts were not included in
the synthesis nor XRD analysis. It is also worth noting that based off the TEM images of each
metal oxide synthesis, no fair conclusion can be drawn on activity beyond successfully
dephosphorylating, as the morphologies remain inconsistent and incomparable.
The synthesis of all the metal oxides (vanadium, cobalt, lanthanum, and samarium) all are of a
similar procedure. Each starts with a metal nitrate or metal chloride and are combined with
sodium hydroxide and water. This mixture is stirred for 22 hours at room temperature in air.
After the time passed, each mixture was washed with an ethanol/water mixture and dried at 90oC
for 12 hours in air. Finally, each catalyst was calcinated at 300oC for a further 12 hours to
increase purity of the catalyst. [Figure 6]
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As we can see, the method remains similar. However, the TEM images provide little information
on morphology, and as previously stated, all remain vastly different that it is difficult to provide
conclusive experimental evidence that one metal oxide works better than any of the others
[Figure 6]. Nevertheless, even with lackluster consistency in particles, it was found that each
metal oxide was able to successfully catalyze a dephosphorylation reaction in the p-NPP
solution.
Figure 6: Synthesis scheme of the various metal oxides The x-ray diffraction patterns are an
important characterization technique for identifying the material composition of nanoparticles by
determining the molecular structure of nanocrystals. Data is gathered by using a beam of x-ray to
strike the crystals, where the scattering beam varies in intensity and angles to produce the overall
graph which can be tested with various sources to determine the crystal composition. The data
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gathered for the XRD is from multiple sources for the nanocatalysts tested [V2O5, Co3O4, Sm2O3,
and La2O3]. For each compound tested, we find that the cobalt oxide, vanadium oxide, and
lanthanum oxide, once accommodating for any background noise, have peaks that are very
similar to that found in various sources. The samarium oxide shared similar peaks in some
aspects, but overall, there was too much background noise affecting the XRD scan. This
potentially comes from the nanopowder not being crushed small enough, as the samarium oxide,
formed larger clumps after being calcined.
Figure 7: Transmission electron microscopy of a) Vanadium oxide (V2O5), b) Cobalt oxide
(Co3O4), c) Samarium oxide (Sm2O3), d) and Lanthanum oxide (La2O3)
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Figure 8: X-ray diffraction of metal oxides as confirmation of the composition of a) Samarium
oxide (Sm2O3) b) Lanthanum Oxide (La2O3) c) Cobalt oxide (Co3O4) d) Vanadium oxide (V2O5)
[XRD sources found in literature]23,24,25,26
ii) Results
The dephosphorylation tests were followed under similar conditions. Each of these catalyst
nanoparticles were tested with varying temperatures of ~50C, 250C, 500C, and 850C. For
consistency purposes, the initial p-NPP concentration was 0.2 mg p-NPP / 1 mL deionized water.
Each catalyst was dispersed in water at 3.5 mg catalyst / 1 mL deionized water, and then after
shaking, 1 mL of this solution as added into the p-NPP solution to begin the experiments. A look
at Figure 9 shows that each catalyst was able to separate at least a portion of the phosphate from
the initial solution.
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Figure 9: A) yield of p-NP at 25oC of metal oxides tested in addition to cerium oxide B) and
yield of phosphate at 25oC of metal oxides tested in addition to cerium oxide.
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However, the p-NPP to p-NP + phosphate reaction is on a 1:1 scale, and so theoretically the
molar amount of phosphate cleaved from p-NPP should be equal to the amount of p-NPP
difference. We found that there was not an exact match in terms of p-NPP dissociated and p-
NP/Phosphate yield for all the catalysts, which implies that poisoning of the catalyst surface with
phosphate must be occurring. As shown in Figure 10, each catalyst has a different percentage of
phosphate attached onto the surface. Even though the surface area and morphologies of the metal
oxides were not very consistent, the amount poisoned on the surface is a great indicator for
which catalyst to consider. Vanadium oxide showcases the highest percent of phosphate
poisoning, followed by lanthanum oxide, then samarium oxide and cobalt oxide are similar.
Because the goal is to maximize the yield of phosphate, we decided the best metal oxide to use
was between samarium oxide and cobalt oxide.
Figure 10: Poisoning of phosphate onto the surface of metal oxides from initial reaction.
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IV) Cobalt Oxide
A) Why Cobalt Oxide
Cobalt oxide performed especially well in terms of the conversion of p-NPP and of the minute
poisoning factor. Although it has slightly more poisoning on its surface than the competing
samarium oxide, cobalt oxide was able to dephosphorylate almost double the amount that
samarium was able to.
In addition, there must be some form of catalyst that can act as an artificial enzyme to help push
this reaction forward and cleave the phosphorus-oxygen bond and separate the phosphate. We
know that samarium oxide has potential to influence methane coupling. However, cobalt oxide
has plenty more examples acting as artificial enzymes. Many papers have experimented with
cobalt oxide and have shown that it can be and has been used as an artificial enzyme. One study
finds that cobalt oxide has a significant effect as a catalase as well as a peroxidase, the latter
allowing oxidation of a substrate to hydrogen peroxide 17. Cobalt oxide has also been used as a
peroxidase as well as a superoxide.18 Because of its ability to manipulate and change oxygen,
this made it a clear contender for mimicking an enzyme to cleave a phosphorus-oxygen bond.
B) Cobalt Oxide Morphology study
To draw a further comparison to determine how cobalt oxide dephosphorylates and to what
strength, a good contrast lies in understanding how different morphologies affects
dephosphorylation. Here, we can study how the morphologies, and consequently surface area,
and temperature affects the rate of dephosphorylation.
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We look at different syntheses and morphologies to determine what the best morphology is in
terms of both efficacy and recyclability. Methods for all the syntheses have been chosen with
scalability in mind; in the case a particle works excellently, in the future it may have engineering
applications and as a result, bulk synthesis is a must have.
i. Nanospheres
We begin with the synthesis of cobalt oxide nanospheres. This method is a modified version of
the technique discussed in our cerium oxide paper. The size and morphology were determined by
transmission electron microscope (TEM) [Figure 11]. The simplicity of this hydrothermal
method lies in the room temperature reaction. For future applications in large scale engineering
aspects, this is excellent as the cost for heat is nonexistent in this scenario. The synthesis
procedure starts with 1 mmol of cobalt nitrate hexahydrate (Co(NO3)3 ∙6H2O) and 32 mL of
0.078M NaOH. This mixture is stirred at 25oC in air for 22 hours at 700rpm. After the synthesis
completes, the nanospheres were collected and washed three times with ethanol and DI water. In
between washes, the mixture was centrifuged at 10,000 rpm for 10 minutes and dispersed in
water for future washing/uses. This method synthesizes particles averaging ~20nm cobalt oxide
nanoparticles.
20
Figure 11: A) Transmission electron microscopy of ~20nm cobalt oxide nanospheres. B) X-ray
diffraction of the cobalt oxide nanospheres
ii. Nanocubes
The synthesis of these cobalt oxide nanocubes followed the method developed by Liu et al.19
First, 0.001 mmol of sodium dodecyl sulfate (SDS) and 1 mmol of hydrated cobalt chloride
(CoCl2∙6H2O) was dissolved in 20 mL of deionized water and stirred at 700rpm. Next, 0.5 mmol
sodium borohydride (NaBH4) was added to include a BH4- precursor necessary to facilitate this
reaction. After stirring for 10 minutes, the solution was transferred into a Teflon-lined stainless-
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steel autoclave where a further 13mL of water was added. The autoclave was placed in a furnace
set at 160oC for 12 hours. Once this synthesis is complete, the resulting solution is then washed
with water/ethanol three times and then the particles are placed to dry at 50oC for 12 hours.
Finally, to ensure all the surfactant is removed from the particle, the powder is then calcined at
300oC for 4 hours. The resulting powder formed nanocubes displayed in Figure 12 at a size of
about 30-50 nm across.
Figure 12: A) Transmission electron microscopy of 30-50nm cobalt oxide nanocubes. B) X-ray
diffraction of the cobalt oxide nanospheres
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iii. Nanorods
Synthesis of the cobalt oxide nanorods followed a modified method shown by Wang et al.20
Their method started with 1.34g hydrated cobalt chloride (CoCl2∙6H2O) and 0.06g of urea
(CO(NH2)2) and each individually dissolved in 20mL of deionized water and stirred. The urea
solution was then added dropwise into the cobalt chloride solution and then stirred for a further
10 minutes. This mixture was then transferred into a Teflon-lined stainless-steel autoclave where
it was then sealed and heated to 105oC for 6 hours. The precipitate was then centrifuged, washed
three times with a deionized water/ethanol mixture, then dried in an 85oC oven for 3 hours.
Immediately after, the powder was calcined at 300oC for 4 hours. The resulting powder formed
nanorods displayed in Figure 13 at a size of about 200nm across. From the X-ray diffraction
data, we see that there is also an extra peak at around 2θ = ~38. When looking at the Urea XRD
pattern, we see a prominent spike around the same angle. Therefore we can conclude that not all
the urea was washed away nor calcined.
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Figure 13: A) Transmission electron microscopy of 200nm wide cobalt oxide nanorods. B) X-
ray diffraction of the cobalt oxide nanospheres
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D) Results
Each of the aforementioned nanoparticles were tested under the same conditions, exactly that of
the initial metal oxide test. Each catalyst was tested at 3.5mg catalyst / 1mL water in a 0.2 mg p-
NPP / 1 mL water solution. Samples were taken and centrifuged to stop the catalytic process at
specified time intervals and the resulting supernatant was run through the UV-Vis
spectrophotometer for 310 nm, 400nm, and then treated with the molybdenum blue assay and
recorded the 890 nm wavelength for phosphate concentration. Each of the nanocrystals had
significantly different performances in dephosphorylating p-NPP into its product p-NP. After 4
hours of reaction, the yield of p-NP reached 94.6 ± 3.1, 82.3 ± 6.4, 44.3 ± 9.6, and 58.9 ± 3.4%
for the nanospheres, nanocubes, nanorods, and commercial catalyst respectively. The trends were
not so similar for some nanoparticles with the phosphate yields. After 4 hours, the yield of
phosphate reached 91.55 ± 3.3, 62.22 ± 5.2, 30.75 ± 7.5, and 62.2 ± 4.7% for the nanospheres,
nanocubes, nanorods, and commercial catalyst respectively. The resulting p-NP and phosphate
yields are shown in Figure 14.
25
Figure 14: Experimental yield of various morphologies of cobalt oxide for a) p-NP b) phosphate
These experiments show that in terms of p-NPP conversion, the nanospheres performed the best
with an impressive 94% conversion with a 92% phosphate yield, followed by the nanocubes,
0
10
20
30
40
50
60
70
80
90
100
0 0.5 1 1.5 2 2.5 3 3.5 4 4.5
Yiel
d o
f p
-nP
at
25
C (
%)
Reaction time (hr)
CeO₂
Commercial
Nanosphere
Nanocube
Nanorod
0
10
20
30
40
50
60
70
80
90
100
0 0.5 1 1.5 2 2.5 3 3.5 4 4.5
Yiel
d o
f P
at
25
C (
%)
Time (hr)
Commercial
Nanosphere
Nanorod
Nanocube
CeO₂
B)
26
commercial, and finally the nanorods. The trend is similar in terms of phosphate yield extracted
as well. However, the amount of phosphate lost to poisoning on the catalyst is also an important
factor to consider. We can see the cubes and rods had more poisoning than the sphere and
commercial cobalt oxide [Figure 15]. Due to the idea that the theoretical yield of p-NP should
equal the yield of phosphate, a simple formula can be used to determine the phosphate adsorbed
and poisoning the catalyst as the following:
Phosphate % Adsorbed = (𝐹𝑖𝑛𝑎𝑙 𝑝−𝑁𝑃)−𝐹𝑖𝑛𝑎𝑙 𝑝ℎ𝑜𝑠𝑝ℎ𝑎𝑡𝑒
𝐹𝑖𝑛𝑎𝑙 𝑝−𝑁𝑃∗ 100
This means that an unknown factor is affecting the nanocubes and nanorods to have a higher
affinity for being poisoned than the other morphologies.
Figure 15: The amount of phosphate that poisoned the catalyst, found by the difference between
the yields of p-NP and phosphate.
E) Kinetics
A range of temperatures and time samples were taken to help understand how these catalysts
compare to each other via activation energy. Here, we assume the cobalt oxide reaction follows
first order kinetics. Therefore, we can calculate this by observing the conversion of p-NPP to p-
0
5
10
15
20
25
30
35
Nanosphere Commercial Nanocube Nanorod
Ph
osp
hat
e %
Ad
sorb
ed
27
NP for each temperature and morphology. In order to determine our rate constant, k, we use the
equation:
𝑘𝑡 = ln (𝑛𝑜,𝑝−𝑁𝑃𝑃
𝑛𝑡,𝑝−𝑁𝑃𝑃)
Where t = reaction time (hr)
no, p-NPP = amount of initial p-NPP (mmol)
and nt, p-NPP = amount of p-NPP at time t (mmol)
Because we know the amount of initial moles of p-NPP at the start of each trial (0.2mmol/mL)
we can determine our rate constant k by comparing the final amount of p-NPP at the first
instance the reaction reaches completion. We repeat this for each temperature and morphology to
collect the reaction rate of dephosphorylation. Shown in Figure 16 is a plot of the reaction rate of
dephosphorylation. Shown in Figure 16 is a plot of the reaction rate of dephosphorylation using
cobalt oxide nanospheres at various temperatures as a demonstration. collect the reaction rate of
28
dephosphorylation. Shown in Figure 16 is a plot of the reaction rate of dephosphorylation using
cobalt oxide nanospheres at various temperatures as a demonstration.
Figure 16: Plot of the reaction rate of dephosphorylation using cobalt oxide nanospheres at
various temperatures as a demonstration.
As it is a first order reaction, we can use 1st order kinetics to create an Arrhenius plot to show the
dependence of the rate constant k with respect to the temperature. From the results, it seems that
the cobalt nanoparticles are relatively close to each other at lower temperatures. However, as the
temperature increases, the spread from the nanocrystals shows more variance [Figure 17]. the cobalt
nanoparticles are relatively close to each other at lower temperatures. However, as the temperature increases, the spread from the nanocrystals shows more variance
29
[Figure 17].
Figure 17: Arrhenius plot showing the variance of rate constant k dependent on temperature
Finally, from the Arrhenius plot, we can derive activation energies from the different
morphologies of cobalt oxide nanocrystals. This is done by using the linearized Arrhenius
equation:
ln 𝑘 = −𝐸𝑎
𝑅𝑇+ ln 𝐴
Where k = rate constant
Ea = Activation energy (KJ)
R = gas constant (8.314 J/mol*K)
A = pre-exponential factor
The Arrhenius equation can be fitted to Figure 16 to find all the variables. The rate constant
divided by the temperature in Kelvin (k/T) is directly related to the slope, the temperature is
-1
0
1
2
3
4
5
6
0.0027 0.0029 0.0031 0.0033 0.0035 0.0037
ln (
k)
1/T (K –1)
Nanosphere
Nanocube
Nanorods
Commercial
30
given, and A is simply e to the power of the y-intercept for each catalyst. We simply plug this
into the equation and find activation energy. The activation energies are 41.4 ± 1.4, 46.9 ± 2.5,
47.8 ± 3.1, and 42.8 ± 2.2 KJ/mol for the nanosphere, nanocube, nanorod, and commercial
catalyst respectively [Figure 18].
Figure 18: Activation energies derived for various morphologies of cobalt oxide.
F) Recyclability
One vital characteristic to mark a good catalyst is the recyclability component; i.e. how often the
catalyst can be used without losing significant efficacy. Therefore, to perform a study on the
recyclability of the catalyst, a simple wash and reuse method was used. Essentially, all the
conditions of the prior dephosphorylation test remained the same. A 0.2 mg p-NPP/ 1 mL water
solution was used, and 1 mL of a 3.5 mg catalyst dispersed in 1 mL water was added. A time
interval sample was not taken; instead, only the final p-NP and phosphate measurement was
taken through the UV-Vis spectrophotometer. After each cycle, the catalyst was centrifuged out,
0.0
10.0
20.0
30.0
40.0
50.0
60.0
E a(k
J/m
ol) Nanosphere
Nanocube
Nanorod
Commercial
31
washed with water/ethanol 3 times, and then dispersed in water and added to the 10 mL of the
0.2 mg p-NPP / 1mL water mixture. This method was repeated 3 times to test how effective the
catalyst was after each use. The results of the phosphate yield show there was a relatively
negligible loss of efficacy in the nanospheres, with approximate a 3% drop each run. The other
morphologies had a varying amount, between 12-20% drop in phosphate yield per run [Figure
19].
It might be notable that the phosphate yield is potentially a combination of recyclability loss as
well as the catalyst releasing the phosphate bound to it. This means that the loss in efficacy on
any recycling attempt could be due to refreshed catalyst poisoning, standard efficacy loss, or a
combination of both. It can be inferred based on the commercial and nanocube morphologies that
it is likely both, as the commercial had relatively little initial poisoning, whereas the nanocube
had a much higher rate. By the 4th run, both nanocube and commercial had approximately the
same phosphate yield, although this cannot be confirmed until a phosphate release study is done
to fully flush all the poisoned phosphate on the catalyst.
32
Figure 19: a) Recyclability of catalysts shown by the amount of phosphate yield after each run,
b) and a graph displaying the average percentage difference in phosphate yields after all the runs.
0
10
20
30
40
50
60
70
80
90
100
0 1 2 3 4 5
Ph
osp
hat
e yi
eld
(%
)
Reaction number
Nanosphere
Commercial
Nanocube
Nanorod
33
V) Conclusion/Future Works
A) Results Summary
We were able to successfully synthesize and test various metal oxides to dephosphorylate the
model reactant. Vanadium oxide yielded the most catalytic poisoning however, at a significantly
higher rate than the others. Even with this in mind, the activation energy trend showed that cobalt
oxide, with around 57 KJ/mol had the lowest, followed by vanadium oxide. This information,
however, does not provide too much conclusive evidence as there is no real connection between
being able to compare the metal oxides as there was no consistency in the particles.
From here, we further determined that cobalt oxide had the best performance and as a result, we
synthesized multiple morphologies to test the catalytic capabilities at various temperatures.
Experiments determined that the cobalt oxide nanoparticles not only performed the best with
approximately a 92% conversion rate, but it also had very little catalytic poisoning onto its
surface with only about a 3% poisoning factor. Additionally, we see that the nanospheres
recyclability factor runs at about 97% efficacy compared to prior runs with reusing the same
nanospheres. However, all the other cobalt oxide nanocrystals were successful in
dephosphorylating a reasonable amount of p-NPP. There was about a 5% decay rate with
recyclability for these nanocrystals, but whether this is due to pure poisoning, pure catalyst
deterioration, or a mixture of both is unknown. The nanospheres performed both the best in
terms of conversion and recyclability, and on top of that, it had the lowest activation energy of
all the nanocrystals, with about 41.4 KJ/mol. This is followed by the commercial catalyst,
nanocube, and nanorod with 42.8, 46.9, and 47.8 KJ/mol respectively.
34
B) Alternative metal oxide
A further look into the other metal oxides tested (Lanthanum, vanadium, samarium, potentially
titanium and iron oxides) and extra studies on surface characterizations could improve the
understanding on what factors contribute to facilitating the dephosphorylation mechanism using
catalysts. Additionally, focusing on different metal oxides and their surface properties could also
shed some light on what affects phosphate poisoning onto surfaces, and what factors of metal
oxide as well as morphology contributes to potency of the poisoning.
C) Engineering Significance
A reliable catalyst always will have room for significant improvement on any process we
currently have in the world. In terms of these cobalt oxide nanospheres, we can see that their
effect on dephosphorylating our p-NPP model reactant was a huge success. This opens the realm
for potential use in many areas. As stated before, a huge problem with large lakes and rivers are
the amount of phosphorus pollution from nearby farms flowing into the tributary rivers. This
causes eutrophication and algal blooms. One method of preventing this would be implementing a
dam with method of purifying the phosphate from the water, as a sort of massive filtration
system. This would allow phosphates from agricultural runoff as well as excess manure/fertilizer
to be captured and reused, as well as acting as a preventative measure for spikes in algal blooms.
While this is not significant in terms of engineering efficiency, it may be for environmental
purposes. One other use could be implementation in wastewater treatment plants as another
method of capturing and filtering. We have recently released a paper discussing the use of using
copper-substituted ZSM-5 in order to recover inorganic phosphorus using anion exchange.21
These have successfully recovered the phosphorus nutrients from wastewater. These zeolites can
35
be used in conjunction with our cobalt oxide to potentially pair the capture and release of the
phosphate ions. The cobalt oxide could be used at the beginning of the wastewater treatment to
capture plenty of the phosphate from the masses. This can be separated and captured with the
zeolites to adsorb as much of the phosphate before the waste can move on to further processing.
Although it sounds like a dirty process, much can be done to further reduce the amount of
human-polluted phosphorus and this is a great first step to achieving such a goal. In such an
example, refer to Figure [] to see that an optimal step would be to incorporate the catalyst into
the clarification stage. If incorporated into the primary clarification stage, the nanocatalysts
would be included into the sludge and wasted when filtered out. This secondary clarification
stage is also where all the biomass components are (nucleic acids, phospholipids, etc) which are
all vital sources of phosphorus present in wastewater.
36
Figure 20: Modelled wastewater treatment plant with the intent of incorporating cobalt oxide
nanospheres combined with Cu-ZSM5 to dephosphorylate, capture, and release excess
phosphates in the sludge.22
D) Improvements
As mentioned regarding recycling, one thing to look at could be understanding how much
surface poisoning factors into the efficacy drop. This can be done by extracting all the
phosphates from the surface of the cobalt oxide nanoparticles to ensure it is as clean of a surface
as possible. Then from there, testing and comparing the new recyclability and phosphate loss
would have a more accurate and specific comparison to how the recyclability is affected from
both new catalytic poisoning and normal efficacy loss. Additionally, a more focused method of
gathering surface area for the nanocatalysts would improve the accuracy of the surface area
comparison, as just rough conclusions were drawn through visual assumptions on the surface
area exposure. One suggestion could be a Brunauer-Emmett-Teller (BET) analysis for more
accurate surface area averages of the cobalt oxide nanoparticles. Further characteristics can be
done using oxygen-temperature programmed desorption (O2-TPD) to determine the strength of
the oxygen vacancies present on each morphology.
Finally, as mentioned just prior, a study on how effective these nanocrystals work in a constantly
refreshing phosphate source would help understand any potential implementations of this
technology and its efficacy over time as a viable counteract to phosphorus pollution.
37
REFERENCES
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(2016)
2: Cordell, D., Drangert, J., White, S., The story of phosphorus: Global food security and food
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3: www.epa.gov/nutrientpollution/problem
4: Patel, J., Parshina-Kottas Y., “Miles of Algae Covering Lake Erie”. NYTimes.com, New York
Times. 2017. Retrieved 2018-05-02
5: Metson et al., Feeding the Corn Belt: Opportunities for phosphorus recycling in U.S.
agriculture. Science of the Total Environment. (2015)
6: Park, J., Craggs, R., Shilton, A., Wastewater treatment high rate algal ponds for biofuel
production. Bioresource Technology (2010)
7: "4-Nitrophenol CAS 100-02-7 | 106798". www.merckmillipore.com. Retrieved 2018-05-02.
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9: Crouch, S., Malmstadt, H., A Mechanistic Investigation of Molybdenum Blue Method for
Determination of Phosphate. Analytical Chemistry. (1967)
10: Kuah, E., et al, Enzyme Mimics: Advances and Applications. ChemPubSoc Europe. (2016)
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11: Capitán, M., Centeno, M., Muñoz-Páez, A., Carrizosa, I., and Odriozola, J., Sm2O3/Al2O3
Catalysts for Methane Coupling. Influence of the Structure of Surface Sm-Al-O Phases on the
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CeO2(111). Catalysis Today. (2018)
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rare earth oxides: Current state of understanding and remaining challenges. Surface Science
Reports. (2007)
14: M. Yashiro, A. Ishikubo, M. Komiyama, Dinuclear Lanthanum(III) Complex for Efficient
Hydrolysis of RNA. Journal of Biochemistry. 1996
15: Shekar, C., Rao, S., Babu, H., Dielectric Properties of Vacuum Deposited Samarium Oxide
Sandwich Structures, Crystal Res. & Technol. (1984)
16: Xu, L., et al., Plasma-Engraved Co3O4 Nanosheets with Oxygen Vacancies and High Surface
Area for the Oxygen Evolution Reaction, Angewandte Chemie. (2016)
17: Mu, J., Zhang, L., Zhao, M., Wang, Y., Catalase Mimic Property of Co3O4 Nanomaterials
with Different Morphology and Its Application as a Calcium Sensor. ACS Applied Material and
Interfaces. (2014)
18: Dong, J., et al., Co3O4 Nanoparticles with Multi-Enzyme Activities and Their Application in
Immunohistochemical Assay. ACS, Applied Materials & Interfaces (2014)
19: Liu, X., Qiu, G., Li, X., Shape-controlled Synthesis and Properties of Uniform Spinel Cobalt
Oxide Nanocubes. Nanotechnologies 16 3035 (2005).
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20: Wang, G., et al. Hydrothermal synthesis and Optical, Magnetic, and Supercapacitance
Properties of Nanoporous Cobalt Oxide Nanorods. Journal of Physical Chemistry. (2009)
21: Manto, M.J., et al., Recovery of Inorganic Phosphorus Using Copper-Substituted ZSM-5.
ACS Sustainable Chemistry & Engineering. (2017)
22: http://empoweringpumps.com/ksb-wastewater-treatment-process, Retrieved 2018-05-12
23: Almoabadi, A., et al., Subzero Temperature Dip-Coating of Sol-Gel Vanadium Pentoxide:
Effect of the Deposition Temperature on the Film Structure, Morphology, and Electrochromic
Properties, Journal of Nanomaterials. (2016)
24: Guria, A., et al., Tuning the Growth Pattern in 2D Confinement Regime of Sm2O3 and the
Emerging Room Temperature Unusual Superparamagnetism, Scientific Reports (2014)
25: Guo, X., et al., New strategy to achieve La2O2CN2:Eu3+ novel luminescent one-
dimensional nanostructures. CrystEngComm. (2014)
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CrystEngComm. (2011)
40
Baltimore, MD
Wilhelm Liano [email protected]
WORK EXPERIENCE
Colgate-Palmolive May 2016
– August 2016
Internship:
Sanford, ME
Ensured the system of production ran smoothly
Troubleshot and ran maintenance or preventative measures for errors in machinery.
Redeveloped and implemented a pathway for mixer to bottling process for stability and
consistency.
o Worked alongside the main design engineer team
o Decreased the error by 0.2% in my time there
Johns Hopkins University October
2017 – Present
Graduate Assistant, Diversity and Inclusion Department
Baltimore, MD
Leading workshops designed to introduce students to the culture at Johns Hopkins and
influence students to be more empathetic and accepting
o Has given me plenty of experience in speaking comfortably in front of large crowds
Working with the team and improve on workshop; co-facilitate events on campus
Johns Hopkins University January
2015 - Present
Researcher
Baltimore, MD
Working on nanoparticle catalyst to develop methods for dephosphorylating molecules where I
have:
o Increased recyclability to 80-90%
o Optimized synthesis methods for consistency
o Captured and redistributed phosphates and nitrates, as well as other important products,
to suit our purposes.
AWARD AND PAPERS
Provost Undergraduate Research Award
March 2016 o Rewarded for work in Dr. Wang’s lab regarding catalysis and recyclability
Sarah K. Doshner Award April 2017
o An award designating top-level research projects and achievements
Recovery of Inorganic Phosphorus Using Copper-Substituted ZSM-5 o ACS, 2017
Recovery of Ammonium from Aqueous Solutions using ZSM-5 o Chemosphere, 2018
41
EDUCATION
Johns Hopkins University
May 2018
MS, Chemical and Biomolecular Engineering
Baltimore, MD
Attended conferences/meetings, collaborated with multiple schools to get projects done
Johns Hopkins University
May 2017
BS, Chemical and Biomolecular Engineering
Baltimore, MD
SKILLS & INTERESTS
Skills: Public speaking, Java, Microsoft Office software, quick adaptability, situation analysis,
strategic planning, negotiations and management. TEM, SEM, XRD, GC-MS trained; proficient
at Matlab, AspenTech, CADPro, VBA.