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-

3

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

4

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

5

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

6

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

9

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]

12

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

13

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)

14

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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.

19

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.

23

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

1: Mayer, B. et al., Total Value of Phosphorus Recovery. Environmental Science and Technology

(2016)

2: Cordell, D., Drangert, J., White, S., The story of phosphorus: Global food security and food

for thought. Global Environment Change (2009)

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.

8: Manto, M., et al, Catalytic Dephosphorylation Using Ceria Nanocrystals. ACS Catalysis.

(2017)

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

Reactivity. Journal of Physical Chemistry (1997)

12: Zhao, C., Xu, Y., Theoretical investigation of dephosphorylation of phosphate monoesters on

CeO2(111). Catalysis Today. (2018)

13: Ganduglia-Priovano, M., Hofmann, A., Sauer, J., Oxygen vacancies in transition metal and

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).

39

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)

26: Xu, C., Controllable synthesis of triangle taper-like cobalt hydroxide and cobalt oxide.

CrystEngComm. (2011)

40

Baltimore, MD

Wilhelm Liano wliano1@jhu.edu

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.