A BIOENGINEERED APPROACH FOR SUSTAINABLE CRUDE OIL POLLUTION

TREATMENT VIA ENTRAPMENT, DISPERSAL AND REMOVAL USING NANO-

CARBOSCAVENGER

BY

ENRIQUE ALEJANDRO DAZA

THESIS

Submitted in partial fulfillment of the requirements

for the degree of Master of Science in Bioengineering

in the Graduate College of the

University of Illinois at Urbana-Champaign, 2015

Urbana, Illinois

Adviser:

Professor Dipanjan Pan

ii

ABSTRACT

Aqueous petroleum contamination continues to threaten life-dependent bodies of water

and surrounding ecosystems including human habitation. It is now well established that most

methods of petroleum remediation are either inefficient or involve highly toxic chemical

dispersants. In this work we adopt a commercially-amenable nanotechnology based approach to

design tunable, biodegradable particles for efficient and safe petroleum remediation.

Exhaustively characterized 100 nm sized hybrid dual-shelled polymeric (PS275-b-PAA50)

and carbon-cored nanoarchitectures (Nano-CarboScavengers or NCS) were derived

predominantly from natural carbohydrate sources and designed for a multipurpose oil

sequestration and dispersion agent that is environmentally undisruptive. NCS distribution on

crude oil contaminated water was found to combine both absorption and dispersion mechanisms

to treat a maximum of 80% of the pollution, while NCS distribution on a petroleum distillate

mixture efficiently treated 91% of the pollution by absorption alone.

NCS exposure to Zebrafish, Vibrio fischeri, and MCF-7 were performed in vitro and

were found to be remarkably un‐inhibitive with LD50: 8 g/L, EC50: 1.28 g/L and IC50: 4 g/L

respectively. This far exceeds expected NCS exposure (ENE) of 1x10-5

g/L in bodies of water.

Finally, NCS degraded in the presence of human myeloperoxidase (HMPO) and horseradish

peroxidase (HRP), suggesting any incidental biological uptake can be enzymatically digested in

living systems.

iii

Dedicated to my Brother and Fellow Engineer

Brandon Clore

iv

ACKNOWLEDGMENTS

This project would not have been possible without the support of many important and

influential individuals. Many thanks to my adviser, Prof. Dipanjan Pan, who guided my work

and gave me the support I needed to continue powering through this project. Also, thanks to my

project collaborators Dr. Santosh Misra, Nicholas Kolmodin, and Christine Promisel as well as

the rest of the Pan Lab Research Group (MatMed Team) at the University of Illinois, Urbana-

Champaign. Thank you to the University of Illinois Graduate College Fellowship, the Support

for Under-Represented Groups in Engineering (SURGE) Fellowship, and the Institute for

Sustainability Energy and Environment for funding my research endeavors. And finally, thanks

to my mother, my fathers, my sister, my brother, my close friends, and endless caffeine who

offered support through such a challenging feat.

v

TABLE OF CONTENTS

CHAPTER 1: INTRODUCTION ………………………...………………………………………1

CHAPTER 2: CURRENT STATE-OF-THE-ART……………………………………..…….…..4

CHAPTER 3: RESULTS AND DISCUSSION…………………………………………...……..10

CHAPTER 4: CONCLUSION…………………………………………………..………………36

METHODS………………………………………………………………………………………40

REFERENCES………………………………………………………………………………..…48

1

CHAPTER 1

INTRODUCTION

1.1 AN OVERVIEW OF PETROLEUM SPILLS

While petroleum is a necessity in today’s modern world1, its production is not free of

harm. As the demand for petroleum continues to rise, we will see a rise in petroleum

transportation traffic2,3

which will inevitably lead to an increase in the possibility of aqueous

crude oil spills, land spills, and fracking byproduct based pollution.1,4–12

An example was

recently seen with the May 19th

, 2015 Santa Barbara oil spill13

. Oceanic spills in 2014 recorded

a total of 4,000 tons of crude oil lost to the environment14

with cleanup costs ranging from

USD12 million to billions6 due to the complex hydrocarbon makeup of various petroleum

grades, sulfur content, and insoluble dirt15,16

which makes the remediation process difficult.

Many manual large scale recovery tactics, such as skimmers paired with booms and suction,

partially alleviate the issue, but leave residual ecologically hazardous hydrocarbon layers on the

micrometer scale17

. This layer is sufficient to prevent sun rays from penetrating a bulk body of

water and hindering O2 mixing, causing a drastic decrease in respiration and photosynthesis in

aquatic ecosystem5,7

. This hindrance means these types of disasters will continue to cause

environmental harm to flora and fauna‐reliant ecosystems even post mass remediation.

2

1.2 DISPERSANTS

Chemical dispersants are a widely adopted substitute used today to treat aqueous oil spills

in massive quantities which do not leave the aforementioned residual layers of floating

petroleum18,19

. Dispersants are made from a cocktail of chemical surfactants including

emulsifiers and detergents used to lower the surface tension between the petroleum and the water

interface20

. This approach is combined with turbulent wave motion to envelop petroleum into

micrometer sized beads, which can then diffuse into the water column21

(Schematic 1). The

process is essential to stop the surface spread of petroleum from reaching beaches and marine

life or becoming susceptible to igniting20

.

Schematic 1: Schematic representation of the mechanism of dispersants on crude oil.

1

2

3

Oil Slick

Dispersant

Surfactant

Interface Hydrophilic

Hydrophobic

Oil

3

1.2.1 Bacterial Bioremediation Aided by Dispersants

Dispersing petroleum into the water column may seem like a poor alternative, but there

are many strains of petroleum digesting microorganisms, such as Alcanivorax borkumensis22

and

Thalassolituus oleivorans23

, that can turn the pollutant into natural byproducts24–28

. Typically

hydrocarbon-degrading bacteria compose 1% of environmental microbiome, but at the first sign

of crude oil leakage into soil or aquatic environments these organisms exponentially grow up to

10% of the bacterial population24

. At first, the plume of crude oil forms emulsions of large

droplets which take a long time to digest by bacteria due to low surface area to volume ratios, but

dispersed hydrocarbons droplets have reduced water interfacial tension and thus form smaller

droplets for faster digestion. Most strains of hydrocarbon degrading bacteria create their own

bio-surfactants, however not in volumes necessary to disperse large spills. Thus, the ability to

disperse petroleum can yield positive results.

1.2.2 Problems Stemming from Dispersants

Unfortunately, studies have shown that the chemicals used for dispersion cause a

synergistically toxic emulsion as found by many studies29–35

. In 2010, 7 million liters of Corexit

EC9500A, a brand named dispersant, was used at the Deepwater Horizon Oil Spill well head29

.

This raised ethical issues about its use in such large quantities, let alone treated directly at the

well head, and sparked many toxicity studies thus exposing its environmental hazards. Protesters

in Santa Barbara have emphasized the dangers of treating the current spill with chemical

dispersants, but the option has not yet been ruled out36

. Despite the harm caused by chemical

dispersants, the mechanism of crude oil dispersion can be applied towards designing an efficient

and safer alternative.

4

CHAPTER 2

CURRENT STATE-OF-THE-ART

2.1 LARGE SCALE REMEDIATION

As discussed earlier, any kind of petroleum spill can become very costly to clean up due

to resources, machines, and workers, combined with the need to remove pollutants quickly to

prevent spread and wild life contamination6. The first response to an oil spill in a large body of

water is to drag large booms, a floating barrier, across the polluted sites between two boats or

skimmers. This collected petroleum is then scooped or vacuumed out of the water and processed

for industrial use. Any residual petroleum can be chemically dispersed or left for bioremediation.

If the petroleum reaches the shore or originates at a land based well or fracking site, then the

common method for cleanup is to simply scoop the sand or dirt away from inhabited sites and

stored where it cannot pollute ground water37

.

New technologies are being proposed constantly for cheaper, quicker, and more efficient

oil spill remediation. Many porous sorbent materials38–42

, sponges43–47

and meshes48–51

(Table 1)

are used to manually separate the hydrocarbons from waters. Some of these materials have

achieved highly efficient loading capacities and reusability with high durability during use in

harsh environments. Unfortunately, these technologies must be used manually and are not yet

suitable for cleaning up such large volumes of crude oil.

5

2.2 IMPORTANCE OF HYDROPHOBICITY

Apart from large scale treatments and chemical remediation, nanotechnology based

materials have been considered as appropriate substitutes. As such, many novel petroleum

extraction nanomaterials with varying mechanisms exist. This list includes gels52–55

,

nanoparticles56

, nanowires41,57

, and magnetic composites58–61

(Table 1).

Almost all of the previously mentioned technologies are created with a theme designed

around hydrophobicity as a main characteristic. The reason being is that petroleum based

pollution is highly insoluble in water and thus oil absorbing materials must carry hydrophobic

attributes for efficient interactions of target hydrocarbon contaminants, especially in polar

environments such as water. In particular, hydrophobic and superhydrophobic materials are the

main focus of recently developed gels55

, iron particles59,61

, and sponges47

for oil based

absorption. Zhu et al47

. recently developed such a sponge with high durability and high oil

absorption capabilities. The study introduced a super hydrophobic polysiloxane coating to a

porous polyurethane sponge and successfully absorbed purified oils. Although materials such as

these are efficient in vitro, they do not take into account the various compositions and

miscellaneous compounds found in petroleum spills such as dirt, sulfur and the variable chain

length and complexity of hydrocarbons15,16,62–66

. Additionally, many fabrication materials and

methods become costly barriers to commercial translation and are thus hindering its possibility

for oil spill remediation.

6

2.3 NANOSHELLED MATERIALS

A particularly novel technique incorporated into nanomaterials used for oil sequestration

is the use of amphiphilic hybrid core/shell structures which can add additional features such as

aqueous dispersion59–61,67,68

. Wang et al61

. entrapped negatively charged iron microparticles

within micellar arrays and used this nanomaterial for soil washing and removing hydrophobic

organic compounds from media. This particle was very efficient in attracting the tested oils;

however its loading capacity was low. A more sophisticated shelled nanoparticle tested for

crude oil treatment is a self-assembled magnetic/polymer hybrid, which uses cross-linked

superhydrophobic iron oxide nanoparticles for hydrocarbon entrapment or weathered crude oil

and subsequent magnetic recovery. Although these materials have improved nanomaterial

functionality with the introduction of a core/shell structure, cost and fabrication barriers exist.

Additionally, only weathered crude oil (crude oil where corresponding lighter distillate

compositions are allowed to evaporate) was tested in this study, so smaller and lighter chain

interactions were not investigated. In all nanomaterials, such as the previous two examples,

where powder distribution is the method for treatment we must assume a less than 100%

recovery of nanoparticles used and thus quantify its potential environmental impact given that

previous studies have shown hazardous fallout from nanomaterials in ecological habitats69–72

.

This aspect is consistently overlooked with many materials awaiting commercial

translation which emphasizes the need for separation from current toxic substitutes such as

chemical dispersants. Thus, these concerns pose a challenge for the makeup of a nontoxic, cheap

and commercially viable product.

7

Material Scale Mechanism Reference

Porous materials Macro Porous Sorption 38–42

Sponges Macro Absorption 43–47

Meshes Macro, Micro, Nano Filtration 48–51

Gels Macro, Micro Sorption 52–55

Nanoparticles Nano Absorption, Filtration 56

Nanowire Nano Absorption, Filtration 41,57

Magnetic

Composites

Micro, Nano Absorption 58–61

Table 1: List of various macro- and nano- materials used for petroleum pollution

remediation.

8

2.4 THE CURRENT UNMET NEED AND PROPOSED SOLUTION

With such a diverse and fragile ecosystem a clear unmet need is to seek an ecologically

safe and efficient material designed for petroleum treatment. Towards this aim we propose a

nanotechnology-enabled carbon-cored nano-architecture derived predominantly from natural

carbohydrate sources for minimal environmental disruption and an amphiphilic dual shelled

approach for a multipurpose oil sequestration and dispersion agent.

2.4.1 Design

In order to effectively treat petroleum contaminated bodies of water with nanoparticle

technology, we must evaluate the requisites to target such hydrocarbon based pollution on the

molecular level, while keeping environmental impact as a priority. A carbon core with a

hydrophobic/amphiphilic dual-shelled structure was designed (Schematic 2) with required

characteristics for petroleum treatment of various grades, compositions, and distillate varieties.

Recovery of waste by booms after treatment is an essential part to keeping an undisturbed

environment, thus buoyancy and accumulation of particle and its insolubility within polar

environments was considered when choosing materials. Not all of the components of crude oil

can be absorbed within a nanoarchitecture framework, thus the entire particle was also designed

to also act as a dispersant to remove additional remnants of un-absorbed pollutant from the

surface of the water and to counteract any residual hydrocarbon micrometer layers from forming.

To the best of our knowledge a dry form powdered nanodispersant has not yet been explored.

Thus we were motivated to create the first multipurpose solid state/powdered material for crude

oil dispersion by bioengineering the framework of the particle.

9

Schematic 2: Graphical representation of the preparation and treatment of the Nano-

CarboScavenger. Nectar agave is used as the main carbon source to produce a carbon

nanoparticle core. Pentacosadiynoic acid is then thermally crosslinked to the carbon core and

coated with a self-assembling amphiphilic block polymer. The NCS can then be distributed

across oil contaminated surfaces in dried powder form where long chain hydrocarbons are

immediately dispersed into water column and short chained hydrocarbons are absorbed within

NCS. The block length shown has representative numbers, whereas the block length used in

this study is PS275-b-PAA50.

10

CHAPTER 3

RESULTS AND DISCUSSION

3.1 SYNTHESIS AND CHARACTERIZATION

3.1.1 Core and Inner Shell

The carbon core was derived from nectar agave, a bio-based starting material, which is a

common sweetener composed of mainly fructose and other carbohydrate sources73

. This basis

allows for an easily modifiable surface for further addition of functional components, as studied

by Mukherjee et al.74

. As previously mentioned, the hydrophobicity of the entire particle is

necessary to drive absorption for any surrounding complex insoluble hydrocarbons that can be

found in petroleum. Thus, the initial shell surrounding the carbon core must be composed of a

hydrophobic residue. A fatty acid 10, 12-Pentacosadyinoic acid (PCDA) was selected to

compose the initial shell not only for its hydrophobic nature, but also for it’s more common use

as a crosslinker75–77

. PCDA’s crosslinking ability has been characterized as

photopolymerization78–80

, using light energy for polymer bond formation, but its thermal

crosslinking ability has not been studied in depth to the best of our knowledge. The synthesis of

carbon core began by heating agave and water with PCDA until all water evaporated, and we

predicted a stable functionalization of PCDA during particle formation. To analyze PCDA

crosslinking, samples were extracted at several time points during hydrothermal synthesis and

measured using Ultraviolet-visible spectroscopy (UV-VIS) (Figure 1). UV-VIS is a fundamental

technique used to measure absorbance or reflectance across a wavelength gradient in the

11

ultraviolet and visible light spectrum. The increase in absorbance peak intensities indicates

polymerization of PCDA and thus successful crosslinking. Particles are then suspended in

tetrahydrofuran (THF) due to newly acquired hydrophobicity and in preparation for further

functionalization.

Dynamic Light Scattering (DLS) is used to measure size of nanoparticles by using

monochromatic laser light scattering of particles in solution with respect to Brownian motion and

time. The laser light scattering intensity fluctuates with destructive or constructive interference

from surrounding particles which can be analyzed by an autocorrelation function to explain size

on a nano-scale. Thus DLS measurements indicated PCDA shelled carbon cores averaged a

diameter of 8 ± 3 nm with a poly dispersity index (PDI) of 0.39 ± 0.01 (Figure 2). Unfortunately,

this organic suspension made anhydrous state characterization techniques difficult to perform but

suspended techniques such as DLS and UV-VIS more viable.

Figure 1: UV-VIS spectrophotometer absorbance measurements of PCDA thermally

crosslinking on to carbon core.

12

3.1.2 Outer Shell Synthesis and Characterization

The amphiphilic diblock copolymer Poly(Styrene)281-block-Poly(Acrylic Acid)49 (PS275-

b-PAA50) was chosen to compose the outermost shell for its ideal hydrophobic polystyrene block

that would aid self-assembly onto the previously synthesized single shelled hydrophobic carbon

core. The self-assembly of this amphiphilic polymer has been thoroughly studied before81–89

and

thus we assume a uniform coating which was further characterized as discussed below.

Additionally, the long styrene chain serves to increase loading capacity and expansion potential

for hydrocarbon absorption and the low density of styrene also adds to NCS buoyancy needed

for easy recovery and polar insolubility. The residual acrylic acid groups are the source for

dispersion of aliphatic groups that are too large or complex for absorption as is found in

unrefined crude oil62,64

. This amphiphilic group also aids in interactions with subsurface dwelling

petroleum layers within a shallow water column90

.

The polymeric self-assembly of the second shell is performed in an organic (THF) to

polar (carbon filtered water) solution titration of polymer and hydrophobic carbon cores. The

residual THF is removed by rotary evaporation to create particle suspension in a purely polar

solvent. The suspension was measured by DLS to monitor size increase and it was found that

particles grew to 115 ± 25 nm in hydrodynamic diameter (Figure 2).

13

For functional evaluation purposes the NCS suspension was dried in a vacuum desiccator

for four hours and collected for further characterization and petroleum treatment. Further

characterization of the dual-shelled hybrid carbon-cored nano architectures involved various

techniques for a thorough understanding of NCS composition, shape, and surface chemistries.

After hydrodynamic size has been established through DLS, transmission electron microscopy

(TEM) was used to assess anhydrous state morphologies and shape to determine characteristics

of the shells.

TEM exposes samples to a high voltage electron beam and captures the intensity of

electrons transmitted through the sample for a high resolution image at a nano scale. Thus, a

Figure 2: Overlaid number averaged hydrodynamic diameter DLS histograms of NCS in

water and PCDA-coated carbon core measured in THF.

14

TEM image of the NCS (JEOL 2100 Cryo TEM, Gatan UltraScan 2k × 2k CCD) gave a clear

depiction of the shells, which appeared as rings and dense carbon core (Figure 3). The styrene

layer of the block co-polymer coating appears as a bright white ring due to the lower density

followed by a darker outer ring being the slightly denser acrylic acid residues. Additionally,

ImageJ analysis was performed on TEM images as a method of sizing dried particle and

produced diameters (Dah) of 109 ± 21 nm. For TEM measurements, 146 individual particles were

counted out of 5 images.

Tapping mode Atomic Force Microscopy (AFM) is an additional characterization

technique that uses a laser reflecting off of a cantilever to measure in the z axis by tapping the

samples with a micron sized tip oscillating at frequency controlled by piezoelectric material.

Thus, AFM was used to characterize anhydrous state morphologies and height of the NCS

particles. AFM images depict a spherical morphology (Figure 4) with average height (Hav)

measured at 111 ± 25 nm. The TEM and AFM size analyses were found to be slightly smaller

than the hydrodynamic diameter (Dav, DLS), presumably due to the flattening of the bushy

polymer chains on the TEM/AFM grids and attributed to the loss of hydrodynamic size during

the dehydration process.

Scanning Electron Microscopy (SEM) is used to measure electron scattering off of

samples through low voltage electron beam exposure and thus is a great technique for visualizing

surface topography of NCS particles. Images were taken after gold sputtering of samples for 30

seconds to increase contrast, which helped to show spherical morphologies amongst a high

concentration of NCS particles (Figure 5).

15

To ensure the PS275-b-PAA50 truly self-assembled with acrylic acid blocks on the outer

surface, we measured the electrophoretic potential (, zeta) of NCS which predicts the electrical

potential around theparticle surface relative to the potential of the dispersion medium. Zeta

measurements on both the carbon core and NCS showed a high negative surface charge (-60 ± 6

mV to -40 ± 6 mV) (Figure 6) confirming the presence of acrylic acid groups. The increase in

negative surface potential from PCDA-coated carbon cores to NCS was possibly due to the

presence of high block chain length of the styrene groups which has uncharged characteristics. It

is known that a high negative Zeta potential reveals great stability and thus shells are assumed to

stay intact during use91,92

.

Energy-dispersive X-ray spectroscopy (EDX) uses X-ray excitation to analytically

determine elemental composition. EDX spectroscopy on both the NCS and carbon core showed

elemental integrity in both samples with significant increase in the oxygen peak post polymer

coating (Figure 7) which can be explained by the addition of oxygen side groups from the PS275-

b-PAA50. UV-VIS analysis on the component groups of NCS showed an additional peak post

coating (Figure 8), which is characteristic of the added block polymer groups.

16

0.5 μm

0.2 μm

Figure 3: TEM Images of NCS on carbon coated TEM grids. (Left) A high voltage (125 kV) image

of NCS with a insert zoomed in to detail shells. (Right) A lower voltage TEM Image (80 kV)

showing higher densities of NCS particles.

Figure 4: Tapping mode AFM Images of anhydrous NCS on freshly cleaved mica. Scale

bar indicates height intensities in nanometers.

17

Figure 5: Gold Sputtered low voltage (0.5 kV) SEM Images of anhydrous NCS on silicon

in decreasing concentrations from left to right. (Right) showing highly detailed surface

morphology of a single NCS particle.

Figure 6: Overlay of Zeta Measurements from NCS (red) and carbon core (green) in water

with a count rate of 285K counts per second at 12 runs per measurement.

18

Figure 7: EDX elemental analysis of carbon core (left), and NCS (right). Orange arrow

shows oxygen peak in both analysis.

Figure 8: A UV-VIS Spectrum analysis (200 nm to 550 nm) of NCS (red), Carbon core

(blue), and PS275-b-PAA50 (black) denoted as BP for block polymer.

19

Figure 9: Wide angle X-ray scattering (WAXS) and small angle X-ray scattering (SAXS)

studies on carbon core and NCS particles. Representative plots of powder WAXS pattern

from (a) NCS and (b) carbon core and SAXS pattern of (c) NCS and (d) carbon core

particles. Variation in d-spacing patterns with change in surface coating in case of carbon

core particles.

20

The composition of shells was also fortified by x-ray diffraction (XRD) measurements

(Figure 9). An extensive study was performed to obtain diffractogram from NCS and

hydrophobic PCDA-coated carbon particles by wide angle X-ray scattering (WAXS) and small

angle X-ray scattering (SAXS) methods. WAXS and SAXS use wide angle and small angle X-

ray scattering respectively to determine crystalline structure of polymers on sub-nanometer sized

structures. Wide and small angle scattering differ in that SAXS measures the elastic scattering of

X-rays while WAXS measures the Braggs peaks caused by X-ray energy loss through interaction

with sample structure93

. WAXS analysis showed additional crystalline patterns from PCDA-

passivated carbon core particles (Figure 9b) compared to NCS (Figure 9a). Similar patterns were

observed for SAXS patterns of NCS (Figure 9c) and carbon core (Figure 9d). Analysis of WAXS

diffractogram revealed the presence of common layered architecture with d spacing of 0.92, 1.52

and 1.15 nm in NCS and carbon cored particles (Figure 9e). Other layered architectures with d-

spacing of 2.7, 1.77 and 0.663 nm were present in the hydrophobic carbon core particles while

NCS particles showed presence of 2.59 nm. Further investigations of SAXS diffractograms

revealed presence of common layered architecture with d-spacing of 4.65 nm which varied to

5.51 nm for the carbon core while coated NCS showed layered architecture of 27.2 nm d-spacing

(Figure 9f). Variations in d-spacing of layered architectures of NCS and carbon core signify the

changes in surface architecture of NCS particles. Appearance of 27.2 d-spacing in NCS particles

associated with polymer coating with bigger particle size compared to 5.51 nm architecture in

carbon core. This is further confirmed by previously analyzed high voltage TEM images of NCS

(Figure 3) which clearly showed ~30 nm sized polymer shells in the form of light and dark rings.

21

3.2 EFFICIENCY

Our strategy for assessing oil spill treatment efficiency involves treating crude oil and

petroleum distillate polluted water samples with powdered NCS and assessing particle loading

capacity and any residual dispersed carbon signatures within the water column. The process

involved adding crude oil or petroleum distillates to carbon filtered pure water samples, which

were then treated with NCS by powder distribution at varying masses to visualize gradual

treatment performance. Post exposure to NCS, samples were placed on a rocking platform

(VWR) at 5 rpm for two hours to resemble turbulent surface wave movement in large bodies of

water94,95

. In all cases pristine NCS and crude oil absorbed NCS accumulated into a floating

clump for easy recovery post treatment (Figure 10a), while only in the crude oil sample,

dispersed oil stayed suspended in the water column (Figure 10b). Post recovery, the NCS clump

had visually changed size and color depending on the amount of oil absorbed and composition of

petroleum. The container, NCS clump, and residual water went through a thorough mass balance

to measure absorption and dispersion capabilities. First, NCS mass pre and post treatment was

mass balanced to measure any absorbed petroleum. Then, residual water with dispersed and

solubilized contaminants is underwent Total Organic Carbon (TOC) analysis to measure mg per

ml of organic carbon present. Finally, the difference in container mass pre and post use serves as

a control or “treatable oil” versus “total oil”, which combined with previous mass balancing is

also used to factor out any NCS that may have been solubilized in the water column.

22

3.2.1 Crude Oil

The crude oil selected for NCS dispersion and absorption efficiency was a Saudi Arabian

medium-heavy crude oil (2.48% sulfur by weight, API Gravity of 31.1, viscosity at 19 mm2/s at

25 C°) for various reasons. A heavier “sour” crude oil variety is more dense and harder to clean

up than lighter “sweeter” compositions in an aqueous spill6. Additionally, this crude oil has a

higher mixture of complex hydrocarbon chain lengths with compounds such as dirt and sulfur16

.

Thus, the performance on this type of oil can only improve with lighter “sweeter” crude oil

varieties.

During the treatment procedure, dispersion effects were immediately apparent (Figure

11) post application of NCS. The NCS crude oil absorption began to take place after extended

interactions during wave action on the rocking platform (VWR). Both absorption and dispersion

Figure 10: (a) Visual treatment of NCS and pellet forming with crude Oil (left) and

Gasoline (right). (b) Crude oil dispersing into water column post treatment with NCS (NCS

removed for visibility).

23

values for each sample were combined in Figure 12a to quantify efficiency results. The graph

showed an interesting trend where the dispersion was dominant at lower masses of NCS used

while absorption was dominant at higher masses of NCS used. We believe the initial dispersion

quantities immediately after NCS application were much higher and were subsequently absorbed

after the two hour wave action incubation period, thus decreasing the dispersed crude oil within

the water column. As we increased the NCS mass in further samples, the total percent of

dispersed crude oil also increased as shown by TOC and an intensity quantification of the

water’s excitation versus emission (EMM) fluorescence spectrum (Figure 13, Table 2). Figure 12

shows the total percent oil treated reached a maximum of 80.4 ± 1.2%.

Figure 11: NCS dispersion property in action on Arabian medium-heavy crude oil.

Dispersion circled in green and aggregate clumping addressed with arrow.

24

(1) (2) (3) (4)

Figure 12: (a) Percentage of crude oil treated by dispersion and absorption. (b) Percent of

total crude oil treated.

250 300 350 400 450 500 550 600 650 700

400

600

800

EM

Wavele

ngth

(nm

)

EX Wavelength (nm)

0.0000.0028870.0057750.0086630.011550.014440.017330.020210.023100.025990.028870.031760.034650.037540.040420.043310.04620

250 300 350 400 450 500 550 600 650 700

400

600

800

EM

Wa

ve

len

gth

(n

m)

EX Wavelength (nm)

0.0000.0028870.0057750.0086630.011550.014440.017330.020210.023100.025990.028870.031760.034650.037540.040420.043310.04620

250 300 350 400 450 500 550 600 650 700

400

600

800

EM

Wa

ve

len

gth

(n

m)

EX Wavelength (nm)

0.0000.0037880.0075750.011360.015150.018940.022730.026510.030300.034090.037870.041660.045450.049240.053030.056810.06060

250 300 350 400 450 500 550 600 650 700

400

600

800

EM

Wavele

ngth

(nm

)

EX Wavelength (nm)

0.0000.0037880.0075750.011360.015150.018940.022730.026510.030300.034090.037870.041660.045450.049240.053030.056810.06060

250 300 350 400 450 500 550 600 650 700

400

600

800

EM

Wa

ve

len

gth

(n

m)

EX Wavelength (nm)

0.0000.0045500.0091000.013650.018200.022750.027300.031850.036400.040950.045500.050050.054600.059150.063700.068250.07280

250 300 350 400 450 500 550 600 650 700

400

600

800

EM

Wa

ve

len

gth

(n

m)

EX Wavelength (nm)

0.0000.0045500.0091000.013650.018200.022750.027300.031850.036400.040950.045500.050050.054600.059150.063700.068250.07280

250 300 350 400 450 500 550 600 650 700

400

600

800

EM

Wavele

ngth

(nm

)

EX Wavelength (nm)

0.0000.0062500.012500.018750.025000.031250.037500.043750.050000.056250.062500.068750.075000.081250.087500.093750.1000

250 300 350 400 450 500 550 600 650 700

400

600

800

EM

Wa

ve

len

gth

(n

m)

EX Wavelength (nm)

0.0000.0062500.012500.018750.025000.031250.037500.043750.050000.056250.062500.068750.075000.081250.087500.093750.1000

Figure 13: EMM Fluorescence images of dispersed crude oil in water with increasing mass

of NCS treatment (left to right). (1) 0 g/L, (2) 1 g/L, (3) 4 g/L, (4) 8 g/L.

25

Figure

13 Image (1) (2) (3) (4)

Area

(a.u.) 1240 3600 5000 5440

Intensity

(a.u.) 336927 1023435 1430561 1530055

3.2.2 Petroleum Distillates

Crude oil can be processed and separated into many gaseous, liquid and solid

compounds66,96,97

such as natural gas, gasoline, lubricating oils, and tar. The various liquid

distillates can pose higher risks in oceanic pollution due to their shorter hydrocarbon chains and

higher solubility in water7,98,99

. Thus a light Louisiana distillate mixture was chosen (0.14%

Sulphur by weight, API gravity 43.2) that included traces of gasoline and naphtha at 32.2 %,

kerosene at 16.3%, gas oil at 11.4%, lubricating distillates at 14.1%, and residuum at 26%.

Post treatment, the absorbed and solubilized quantities of the petroleum distillate were

analyzed (Figure 14a) and unveiled a max loading capacity of 9.9 mg of distillate per mg of NCS

with a max total absorption of treated petroleum at 91.0% (Figure 14b). The higher loading

capacity is thought to come from the lighter and smaller chains present in petroleum distillates

which fit into the polystyrene block segment of the amphiphilic shell. EMM fluorescence

analysis on the treated water also showed a decreasing trend in solubilized hydrocarbon residues

Table 2: Table indicates area and intensity of fluorescent peaks measured by ImageJ in

arbitrary units.

26

which indicates a true removal of contaminants, including subsurface contaminants (Figure 15,

Table 3).

(1) (2) (3) (4)

Figure 14: (a) Loading capacity of NCS measured by mg distillate absorbed per mg of

NCS used, (b) percent of total petroleum distillate treated.

250 300 350 400 450 500 550 600 650 700

400

600

800

EM

Wavele

ngth

(nm

)

EX Wavelength (nm)

0.0000.0061560.012310.018470.024630.030780.036940.043090.049250.055410.061560.067720.073870.080030.086190.092340.09850

Absorbance, 16 mg

250 300 350 400 450 500 550 600 650 700

400

600

800

EM

Wavele

ngth

(nm

)

EX Wavelength (nm)

0.0000.0061560.012310.018470.024630.030780.036940.043090.049250.055410.061560.067720.073870.080030.086190.092340.09850

Absorbance, 16 mg

250 300 350 400 450 500 550 600 650 700

400

600

800

EM

Wavele

ngth

(nm

)

EX Wavelength (nm)

0.0000.0086560.017310.025970.034630.043280.051940.060590.069250.077910.086560.095220.10390.11250.12120.12980.1385

Absorbance, 2 mg

250 300 350 400 450 500 550 600 650 700

400

600

800

EM

Wa

ve

len

gth

(n

m)

EX Wavelength (nm)

0.0000.0086560.017310.025970.034630.043280.051940.060590.069250.077910.086560.095220.10390.11250.12120.12980.1385

Absorbance, 2 mg

250 300 350 400 450 500 550 600 650 700

400

600

800

EM

Wavele

ngth

(nm

)

EX Wavelength (nm)

0.0000.023880.047750.071620.095500.11940.14320.16710.19100.21490.23880.26260.28650.31040.33420.35810.3820

Absorbance, 0.5 mg

250 300 350 400 450 500 550 600 650 700

400

600

800

EM

Wa

ve

len

gth

(n

m)

EX Wavelength (nm)

0.0000.023880.047750.071620.095500.11940.14320.16710.19100.21490.23880.26260.28650.31040.33420.35810.3820

Absorbance, 0.5 mg

250 300 350 400 450 500 550 600 650 700

400

600

800

EM

Wavele

ngth

(nm

)

EX Wavelength (nm)

0.0000.029880.059750.089630.11950.14940.17930.20910.23900.26890.29880.32860.35850.38840.41830.44810.4780

Absorbance control

250 300 350 400 450 500 550 600 650 700

400

600

800

EM

Wa

ve

len

gth

(n

m)

EX Wavelength (nm)

0.0000.029880.059750.089630.11950.14940.17930.20910.23900.26890.29880.32860.35850.38840.41830.44810.4780

Absorbance control

Figure 15: EMM Fluorescence images of solubilized hydrocarbons in water with

increasing mass of NCS treatment (left to right). (1) 0 g/L, (2) 1 g/L, (3) 4 g/L, (4) 16 g/L.

27

Figure

15 Image (1) (2) (3) (4)

Area

(a.u.) 4557 3937 3825 3141

Intensity

(a.u.) 561742 487250 456308 388888

In an effort to further test NCS absorption capabilities of small hydrocarbon chained

petroleum distillates, gasoline was considered because of its high concentration of octane

groups100,101

while being one of the most widely used consumer bought petroleum products102

.

Gasoline was mixed with AmeriColor Blue Oil Dye (Figure 16), a hydrophobic dye, to visualize

any changes on the surface of the sample. The resulting treatment visually demonstrated gasoline

absorption and showed similar clumping as seen with the distillate mixture (Figure 10a).

Hitherto, all samples were tested on carbon purified water, but in order to mimic an

ocean's salinity, a single crude oil, distillate, and gasoline sample experiment were tested on 8 wt

% salt water with the same methods previously mentioned (ratio of 4 mg NCS to 16 mg oil).

Interestingly, this study produced similar results. This is expected since NCS treatment activity

only occurs on the surface of the water and not in the water column.

Table 3: Table indicates area and intensity of fluorescent peaks measured by ImageJ in

arbitrary units.

28

3.3 TOXICITY

As mentioned, the NCS is designed to not only treat petroleum spills with high efficiency

but also to minimize environmental interference. Thus we turn to investigating any toxicity or

detrimental effects across aquatic life, toxin sensitive bacteria, and human cells. To understand

the scale at which any unrecovered NCS dwell in the surrounding environment, we assume an

expected NCS exposure (ENE) calculated to be maximum 1x10-5

g/L in a body of water. As an

initial precaution to test NCS effect on water acidity, an NCS water suspension post fabrication

(roughly 40 g/L) had a pH of 6.2.

In order to grasp the potential solubility NCS in an aqueous environment, an initial TOC

analysis was performed on 100 ml of deionized water tumbled with 50 mg NCS for 12 hours.

After a redundant measurement through mass balance and TOC analysis, a total of 2 mg had

dissolved for a total solubility of 0.02 g/L. For all toxicity treatments, the NCS stayed buoyant on

the surface of the water as it would during actual practice. Thus, any reported EC50, LD50, or

IC50 values calculates combined floating and any solubilized NCS exposed to test water.

Figure 16: Indigo Carmine dye. Mixed with gasoline to visually show absorption by NCS

29

3.3.1 Vibrio Fischeri

Acute bacterial toxicity testing was performed by a Microtox assay. The procedure

utilizes bioluminescent bacteria, Vibrio fischeri, and measures toxicity based on the reduction of

light produced by the bacteria exposed to sample materials. This procedure is a standard method

for acute toxicity testing and is desirable due to high throughput, it’s sensitivity to a wide range

of compounds, and its comparability to other toxicity tests103–105

. Multiple samples were

measured at varying concentrations between 8 g/L and 1 g/L of NCS. Each sample was

subsampled at 0%, 9%, 36%, 63%, and 90% of starting concentration with a 5 minute exposure

time before light measurements were performed. The effective concentration which produces

50% maximum response, EC50, was calculated by linear regression and found to be 1.28 g/L.

3.3.2 Safety profile of NCS in 2D-Human Cell Culture

The protocol of NCS uses leads to possibility of leaving traces of NCS in water body and

nearby areas to reach natural drinking water sources. To evaluate the probable effects of such

NCS traces on human health and particularly human cells, a 2D-culture of MCF-7 was exposed

to various concentrations of NCS. Cytotoxicity was measured by MTT reduction assay using

plate reader (Synergy HT, Bio-Tek). IC50 was calculated from percent cell viability at various

concentrations of both the particle formulations was calculated 8 g/L for polymer coated NCS

while for uncoated carbon cores it was found to be ~1 g/L indicating a huge surge in cell

viability upon surface protection of single shelled carbon core particles. These levels were at

least ~ 2x 104 times to be generally used in oil treatment experiments and the ENE (Fig 17a-f),

while at least 105 times higher than the probable amount reaching to water sources. It revealed

30

the highly safe profile of these particles even on being left in traces around the treated water

body and reaching in very drinking water sources.

31

Figure 17. Morphological and growth density variations in human cell culture. Cell toxicity

experiment was performed in MCF-7 cells, plated at a density of 10,000 cells/well in a 96-

well plate. Cells were incubated for 48 h with NCS and hydrophobic carbon core samples at

concentration of 0.0035 to 4.0 mg/ml. Cell density and morphology of treated cells were

visualized by bright field imaging (a-f). Cell toxicity was quantified by MTT assay (g) and

used for calculating IC50 values (h).

32

3.3.3 Zebrafish Toxicity Assay

Zebrafish (Danio Rerio) was chosen as a model organism for toxicity testing. This

species has shown to reproduce and develop quickly with transparent characteristics for internal

organ analysis only hours post fertilization106–108

. The zebrafish model is also an ideal vertebrate

aquatic organism, thus any toxic exposure to NCS will closely resemble that of a realistic marine

environment. The egg water (growth media) used to culture zebrafish was exposed to various

concentrations of NCS with an exposure time of 24 hours. Five sets of fifteen wild type embryos

26 hours post fertilization were treated with 5 ml of NCS exposed egg water for 24 hours. Issues

such as hatch inhibition, developmental delay, edema, and blood pooling did not begin to show

until ENE was far exceeded (Figure 18a-c). The LD50 was indicated when 50% of embryo

contained severe symptoms and is calculated at 4 g/L (Figure 18c).

33

Figure 18: Zebrafish treated with NCS exposed egg water 26 hours post fertilization at (a)

0 g/L, (b) 1 g/L, (c) 4 g/L. Small eye developmental delay is shown with purple arrow. Lack

of blood in heart is shown with red arrow. Blood pooling is shown with blue arrow. Edema

is shown with green arrow. Scale bar, 0.5 mm.

34

3.4 ENZYME DEGRADATION

The NCS is designed to be buoyant for easy surface recovery but 100% efficiency in

recovering nanoparticles is quite difficult. Thus, we must assume nanoparticle in pristine state

and/or post interaction with petroleum will stray into unwanted territories. As part of this aim to

create a truly safe nanomaterial, we must consider the possibility of direct interaction with

surrounding flora and fauna and any accidental digestion. Hence, we look at plant and human

enzymatic digestion of NCS.

3.4.1 Degradation in presence of Horse Radish Peroxidase (HRP)

Any disruption in aquatic plant life can lead to a disturbance in the rest of the surrounding

ecology since many organisms and herbivores are dependent on plants as a source of food and

shelter, thus we turn to a common plant digestion enzyme horseradish peroxidase (HRP) and

look at its interactions with NCS, specifically at its potential to be degraded. HRP has been

shown to digest single-walled carbon nanotubes109,110

and thus our procedure follows suit. Due to

the slow nature of HRP enzyme activity, 1 mg/ml of NCS was suspended with 0.5mg/ml HRP in

5 ml of Dulbecco’s phosphate buffered solution and 80 μM of hydrogen peroxide and then

statically incubated at 4 C in the dark. Five samples were prepared to measure control and 1, 2,

4, and 8 week time points.

The molecular morphology of NCS is predicted to break down as carbon structures

become oxidized and transitions associated with sp2 bonds of fresh NCS would reduce. To

measure this change samples were drop-casted onto glass slides and analyzed by Raman

spectroscopy observed between 120 and 2700 nm wavelengths which carry the characteristic G

and D bands found on carbon structures111

. After graphical analyses we found a significant

35

decrease within both of these peaks over extended exposure of enzyme activity indicating

degradation leading to sp2 and sp

3 carbon bond oxidation (Figure 19a).

3.4.2 Degradation in Presence of Human Myeloperoxidase

In the case of residual NCS particles emerging in human food sources we must evaluate

degradation by a human digestion enzyme. We investigated the lysosomal enzyme human

myeloperoxidase (HMPO). Our methods involved preparing five suspensions of 1 mg/ml NCS in

Dulbecco’s phosphate buffered solution and 200 μM of hydrogen peroxide. 100 μg of HMPO

enzyme (activity > 50 units/mg Protein, Sigma) was added to four of the five samples and then

statically incubated at 37 C in the dark. Samples were collected at 6, 12, 24, 168 hours and drop

casted on glass slides for Raman analysis (120 to 2700 nm). Similarly to the HRP degradation,

we found that both the G and D band significantly decreased in intensity indicating a dramatic

change in the NCS sp2 and sp3 bond morphology (Figure 19b).

Figure 19: Enzyme degradation of NCS by HRP (a) and HMPO (b) between 120 and 2700

nm. Operated at 2% laser power for 1 min with a 532 nm laser.

36

CHAPTER 4

CONCLUSION

4.1 CONCLUSION

Here we have disclosed a compelling nanoparticle-based approach to oil spill

remediation, which excels in treatment efficiency while maintaining environmental stability.

These highly characterized NCS have the capability to treat a broad spectrum of petroleum and

corresponding distillate based pollution in salt or non-salt aqueous environments. Post powder

based distribution methods, the NCS was found to selectively absorb short chained hydrocarbons

present in many common petroleum distillates with a high loading capacity. When presented to

crude oil contamination, the NCS was found to initially disperse the pollutant rapidly and then

consecutively absorb it after wave action interactions. Additionally, molecular shells and

powdered application were designed for increased handling during large scale oil spill

remediation that lead to material clumping post treatment for simple recovery by nets or booms

without leaving residual signatures in the aqueous environment.

In any oil spill treatment where nanomaterials are used, any unknown consequential

effects must be carefully studied. As a result, we have extensively considered the potential

interactions and effects caused by NCS with biological organisms if recovery is not 100% in

treated areas. Zebrafish was our first model organism tested for the reason that it is a

characteristic vertebrate marine animal. Exposure of zebrafish egg water to different

concentration of NCS showed an LD50 of 4 g/L. The next step was to test V. fischeri, a standard

37

model bacteria used for acute toxicity testing, in order to quantify effects on smaller biological

organisms. Microtox tests at different NCS concentrations for a 24 hour exposure time showed

an EC50 of 8 g/L. An additional toxicity test was performed on the human breast cancer cell line

MCF-7 for details on NCS safety profile in case of mammalian ingestion. Various concentrations

of NCS were exposed to cultured cells and cells were counted 48 h post exposure. The calculated

IC50 was found to be 8 g/L. In all toxicity tests the effective concentration that caused a 50%

toxic effect, either inhibition or death, far exceeded the ENE and thus we can conclude NCS to

be remarkably un‐inhibitive.

The resulting NCS offers a cheap, non-toxic, and easy to produce material for complex

hydrocarbon cleanup in aqueous environments. Overall treatment effectiveness surpass other

nanoparticle based treatments, as confirmed by TOC analysis, fluorescence studies and other

analytical means, making the NCS a superior substitute.

38

4.2 FUTURE AIMS

The continued research in nanotechnology based petroleum spill treatments has great

potential to improve the efficiency and environmental impact of these novel nanomaterials.

There are still many necessary steps that need to be addressed in depth before commercialization

of the NCS, which will further improve its ability to revolutionize the way we treat our

petroleum contamination. The next studies should focus on optimization of particle design,

material, and treatment efficiency. Simulation studies of small chain hydrocarbon interactions

with several candidate block polymers will help identify the best candidate for dispersion and

absorption during treatment of several types or composition of petroleum pollution. The

crosslinker used as the primary shell for adding hydrophobicity to the carbon core should be re-

investigated for maximizing pull on water insoluble material. A stronger hydrophobic or even

oleophilic, interaction would aid in increasing loading capacity per milligram of NCS. Finally,

the source used to make the carbon core should navigate to a material with high nitrogen and

phosphorous content. A particle with this composition can double as a material used for

bioaugmentation. This is a process used to supply extra nutrients to hydrocarbon degrading

bacteria which enhances bioremediation of petroleum micelles. A great candidate for such a

carbon core can be biomass derived carbon particles, such as algae or biochar112

.

Moreover, the recyclable properties of NCS post recovery will be analyzed for reusability

and retrieving excess petroleum. Recovered powdered NCS and petroleum can lead to an overall

increase of waste material. Thus any refining, processing or redistributing of post treated

petroleum will be investigated such as repurposing as tar such as roofing or asphalt, since they

share similar compositions113

.

39

Currently, meshes44,48–50

are highly reusable and highly efficient for separating oil from

water with low oil contamination, thus increasing the possibility to use recovered petroleum for

industrial processing. In future studies, we will attempt to incorporate the NCS into a mesh for

handheld or scale up use.

Finally, we will explore the possible multi-functionality in aqueous pollution treatment

by testing improved NCS against many types of pollutants. This includes petroleum based

pollution such as fracking water waste, land spill contamination, and even crude oil

compromised animals. We would also expand the pollution cleaning capabilities to separating

bacteria, or other microorganisms from drinking water. And finally, we would like to investigate

NCS ability as a chelator for harmful heavy metal contamination in water and in biologicals.

40

METHODS

Unless stated otherwise, all material and reagents were purchased from Sigma–Aldrich, St.

Louis, MO and were used without further purification. Solvents were used as received and

without further purification.

NCS Synthesis:

The carbon core of the NCS was produced by a hydrothermal method of controlled heating a

suspension of 250 mg/ml of nectar agave (HoneyTree’s Organic Agave Nectar, Onsted, MI) and

37.5 mg/ml of pentacosadiynoic Acid (Sigma-Aldrich, St. Louis, MO) in nanopure water (0.2

um) at 250 degrees C° for 45 minutes or until all water evaporates and product turns black mass.

This residue was then soaked in THF overnight then resuspended by sonication (Q700, Qsonica

Sonicators, Newtown, CT) in an ice bath (to prevent evaporation) for 8 minutes at 1 amplitude

with intervals of 5 seconds on and 3 seconds off. The suspension was filtered through a 0.45 µm

and 0.22 µm PTFE membranes. The suspension was dried out to produce a residue. This residue

and (PS275-b-PAA50) at a 10:2 mass ratio respectively, were resuspended in THF. The volume of

THF used is the minimum required to fully dissolve solutes. Water was then added rapidly at

twice the volume of THF to produce the block polymer coated nanoparticles known as the NCS.

This suspension was placed in a vacuum to dry and then scarped/grinded into powder form.

PCDA Crosslinking:

In a 20 mL scintillation vial, agave nectar (HoneyTree’s® Organic Agave Nectar, Onsted, MI)

and 10,12-pentacosadiynoic acid were mixed in water (4 mL) at concentrations of 250 mg/ml

and 37.5 mg/ml, respectively. The sample was bath sonicated (Branson 1510) for 10 minutes at

41

room temperature and then mechanically mixed (Benchmark Bench Vortex Mixer). The

agave/PCDA that did not dissolve was separated from the suspension. The scintillation vial was

then placed on a hot plate at 250 oC and samples were collected at 0, 4, 5, 6, 8, 9, and 10

minutes. The crosslinking was monitored using a UV-VIS Spectroscopy (GENESYS 10S). To

measure the samples collected, a 1 to 20 dilution of the heated sample was prepared in deionized

water for each UV-VIS measurement where a stock solution agave in water at the same

concentration was used as a blank

Efficiency Quantification:

For dispersion quantification of heavy crude oil we used Saudi Arabian medium-heavy crude oil

(2.48% sulfur by weight, API Gravity of 31.1, Viscosity at 19mm2/s at 25

Celsius, ONTA). One

hundred and twenty microliters of the crude oil was pipetted onto 50 ml of carbon filtered water.

The crude oil surface layer was treated with NCS and the container was then placed on a rocking

platform (VWR) at 5 rpm for two hours. The NCS aggregates were removed and weighed while

the water layer was extracted from the crude oil layer for TOC analysis.

For NCS absorption quantification of petroleum distillates, we purchased a Louisiana

distillate sample (0.14% Sulphur by weight, API gravity 43.2, ONTA) with traces of gasoline

and naptha at 32.2 %, kerosine at 16.3%, gas oil at 11.4%, lubricating distillates at 14.1%, and

residuum at 26%. Fifty microliters of the distillate sample was pipetted onto the surface of 10 ml

of water. The sample was then treated with NCS at varying masses. The clump formed by the

CarboScavenger was removed and weighed, while the extracted water was analyzed by TOC.

42

HRP Degradation:

One mg/ml of NCS was suspended with 0.5mg/ml HRP (Thermo Scientific) in 5 ml of

Dulbecco’s phosphate buffered solution and an excess of 80 μM of hydrogen peroxide and then

statically incubated at 4 C in the dark as previously reported

109. At one week, two weeks, four

week, and eight week time points, the suspensions were removed from incubation, centrifuged at

10k RPM for 30 seconds and the supernatant was replaced with Nanopure water to halt any

residual enzyme reaction. The suspension was drop casted onto glass slides for Raman analysis

(120 to 2700 nm at 0.2% laser power for 60 seconds).

HMPO Degradation:

Our methods involved making 5 suspensions of 1 mg/ml NCS in Dulbecco’s phosphate buffered

solution and 200 μM of hydrogen peroxide. One hundred μg of HMPO enzyme (activity > 50

units/mg Protein) was added to four of the five samples and then statically incubated at 37 C in

the dark. Hydrogen peroxide was added every five hours to maintain a 200 uM concentration and

to keep enzyme activity constant as previously reported114

. The treated and control samples were

incubated at 37℃ for 24 hours. At 6, 12, 24, 168 hours, samples were removed from incubation

and placed in 4℃ environment for 4 hours to halt enzyme activity. Samples were then collected

and drop casted on glass slides for Raman analysis (120 to 2700 nm at 2% laser power for 60

seconds).

Total Organic Carbon Analysis:

Total Organic Carbon analysis was performed at the Illinois Sustainable Technology Center.

Carbon measurements were performed with a Shimadzu TOC-V analyzer. The instrument was

43

operated in non-purgeable organic carbon mode with addition of 2M hydrochloric acid and a

sparge time of 1.5 minutes. The instrument was calibrated daily by serial dilutions of a potassium

hydrogen phthalate stock solution over a range of 1 mg/L to 100 mg/L. Three to five injections

were made on each sample with a reproducibility target setting of 0.1 maximum standard

deviation.

Fluorescence Imaging:

Fluorescent measurements were performed at the Illinois Sustainable Technology Center. EEM

measurements were performed with a Horiba Aqualog scanning

spectrophotometer/spectrofluorometer. The samples were scanned over an excitation range of

240 nm to 700 nm and an emission range of 246 nm to 828 nm. The integration time was set at

0.1 seconds and the wavelength scan increments were set for 3nm and 5nm, excitation and

emission respectively. All samples were blank subtracted with data obtained from analysis of a

deionized water blank. Post processing of the fluorescence data included inner-filtering

correction with data obtained by simultaneous absorbance measurements and Rayleigh masking

of signals produced as a result of light scattering of water. In addition, all emission intensities

were normalized to a 1 mg/L quinine sulfate solution that was analyzed prior to each assay.

RAMAN Spectroscopy:

All Raman measurements were taken on a Nanophoton Raman instrument (Materials Research

Building, UIUC) with a 532 nm wavelength laser for one min at 0.2% laser power using a 20x

objective. For each spectrum a grating (600 l mm-1

) scan was taken over the range 120 - 2700

cm-1

. An average of 20 spectra was recorded and averaged for every sample.

44

Dynamic Light Scattering Measurements:

Hydrodynamic diameters were determined using a Malvern Zetasizer ZS90 particle size

analyzer, while scattered light was collected at a fixed angle of 90°. A photomultiplier aperture

of 400 mm was used, with the incident laser power adjusted to obtain a photon counting rate

between 200 and 300 kcps. Measurements for which the measured and calculated baselines of

the intensity autocorrelation function agreed to within +0.1% were used for diameter values.

Hydrodynamic diameter was analyzed using number distribution in accordance with various

previous reports115

. All determinations were made in multiples of 3 consecutive measurements

with 15 runs each.

Determination of Surface Zeta Potential:

Zeta potential (ζ) values for NCS was determined with a nano series Malvern Zetasizer zeta

potential analyzer. Data were acquired in the phase analysis light scattering (PALS) mode

following solution equilibration at 25 °C when calculation of ζ from the measured

electrophoretic mobility (μ) employed the Smoluchowski equation: μ = εζ/η (where ε and η are

the dielectric constant and the absolute viscosity of the medium, respectively). Measurements of

ζ were reproducible to within ± 6 mV of the mean value given by 3 determinations of 15 data

accumulations.

UV-VIS Measurements:

The absorption spectra for NCS and PCDA crosslinking were acquired in a Genesys 10S

UV−VIS Spectrophotometer (Thermo Scientific, Rockford, IL).

45

Transmission electron microscopy measurements

A drop of CarboScavenger suspension was placed on a carbon coated TEM grid. The TEM

images were acquired on a JEOL 2100 Cryo TEM machine and imaged by Gatan UltraScan 2k ×

2k CCD.

Atomic Force Microscopy:

A Digital Instruments Dimension 3000 series AFM (from MRL facility, UIUC) was used for

scanning the samples using standard Veeco tapping mode silicon probes with a platinum–iridium

(PtIr) coating. In a typical experiment, suspended NCS samples were dried in a desiccator on

freshly cleaved mica. Dried samples were placed on the AFM platform and scanned. Pertinent

scanning parameters were fixed as follows: Example of tip velocity: 4 m/s for 2 m, 15 m/s for 5

m, 30 m/s for 10 m; resonant frequency (probe): 60–80 kHz. Aspect ratio: 1:1; resolution: 512

samples/line, 256 lines. 2.10.

Scanning Electron Microscopy and EDX Analysis:

Scanning electron microscopic studies were performed on coated NCS to evaluate surface

properties of their clusters. Additional Energy dispersive X-ray (EDX) spectrum analysis was

also performed to demonstrate the presence of elements in addition to C and O in particle

preparations. Samples were prepared on metal stubs decorated with Si block. Suspension of

particles were used to drop cast on carbon tape and allowed to air-dry before putting in vacuum

for 5 h.

SEM images were taken on Hitachi S-4700 High Resolution SEM machine with

capabilities of high resolution low voltage imaging and connected to cold field emission gun (2.5

46

nm resolution at 1kV, 1.5 nm resolution at 15 kV) while EDX was performed on ISIS EDS X-

ray Microanalysis System (Oxford Instruments) using in build software.

Wide and Small angle X-ray diffraction studies:

WAXS/SAXS measurements were carried out on x-ray diffraction machine with Pilatus 300K

areal detector (172 μm pixel). The Cu Kα radiation (λ = 1.54 Å) was collimated with x-ray

mirror and two pairs of scatter less slits and passed through the evacuated path of 1600 mm from

source of Cu Kα radiation to sample holder. The 2D-patterns were recorded on an image plate

and processed using software. All the measurements were made in the transmission mode. The

sample to detector distance was 1400 mm for SAXS and 136 mm for WAXS, respectively.

Powdered NCS and carbon core samples were used for the analysis.

Zebrafish Toxicity Tests:

Zebrafish toxicity testing was performed at Northwestern University with Professor Jacek

Topczewski. The egg water was exposed to concentrations of NCS (0.5, 1, 2, 4 g/L) for 24 hours.

Five sets of fifteen wild type embryos 26 hours post fertilization were treated with 5 ml of NCS

exposed egg water for 24 hours. Embryo viability were then analyzed under a light microscope

and imaged.

Microtox Assay:

Microtox was performed at the Illinois Sustainable Technology Center. The acute toxicity

reagent, reconstitution solution and diluent were obtained from Modern Water. The phenol stock

material was obtained from Alfa Aesar (99.5% purity) and sodium chloride was obtained from

Fisher Scientific (99.9% purity). Sodium chloride was used to adjust the osmotic pressure of

47

each test sample and reference material to approximately 2% NaCl. Each sample was assayed at

0%, 9%, 36%, 63%, and 90% concentrations. The measurements were performed with an Azur

Environmental M500 Microtox analyzer. The test organism was incubated at 15oC for 10

minutes before exposure. The exposure time was 5 minutes and light measurements were

performed before and after exposure to test materials. The effective concentration which

produces 50% maximum response, EC50, was calculated by linear regression of the difference of

pre-exposure and post exposure light emission versus concentration of test solution. A phenol

reference material was also analyzed with each run as quality control. The known EC50, for

phenol with 5 minute exposure is in the range of 13 to 26 mg/L116

. Therefore for the test to be

considered valid, assay of phenol reference material was required to produce results within this

range.

48

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