Airborne Microbial Decontamination using Dielectric ...blog.novaerus.com/...E-coli-DBD-treatment-report.pdf · Dielectric barrier discharge (DBD) is a popular approach to produce

Novaerus Research Project

Airborne Microbial Decontamination using Dielectric Barrier Discharge Technology

Study Report

(2013-2014)

Submitted to Novaerus Inc.,

Dr. Ram Prasad Gandhiraman,

Principal Investigator Universities Space Research Association

615 National Avenue, Suite 220 Mountain View, CA 94043

Email: [email protected]

Oct 22, 2015

Oct 22, 2015 Page �1

mailto:[email protected]


Aim

The objective of the research is to explore the efficacy of dielectric barrier dis-charge (DBD) technology for deactivating airborne micro-organisms and study the effects of the DBD on surface chemistry and morphology of aerosolized E coli.

Researchers, Advisors and Collaborators who provided valuable scientific comments

Ms. Jaione Romero-Mangado, Microbiologist Science & Technology Corporation NASA Ames Research Center N239 R175 Moffett Field,CA 94035 Phone: 650-604-0323 Email: [email protected]

Dr. Dennis Nordlund, Staff Scientist Stanford Synchrotron Radiation Lightsource SLAC National Accelerator Laboratory Menlo Park, California 94025, USA Email: [email protected]

Dr. M. Meyyappan, Chief Scientist for Exploration Technology NASA Ames Research Center Moffett Field, California 94035, USA Email: [email protected]

Dr. Jessica E. Koehne, Scientist, NASA Ames Research Center Moffett Field, California 94035, USA Email: [email protected]

Oct 22, 2015 Page �2






Dr. David J. Loftus, Scientist, NASA Ames Research Center Moffett Field, California 94035, USA Email: [email protected]

Dr. Stephen Daniels, Senior Lecturer National Center for Plasma Science and Technology, Dublin City University Dublin 9, Ireland Email: [email protected]

Synchrotron X ray study: The high energy synchrotron x ray study was carried out at Stanford Synchrotron Radiation Lightsource, SLAC National Accelerator Laboratory, Menlo Park, California. The proposal titled “Surface Analysis of Bacterial Cell Wall for Airborne Infection Control” was awarded beam time under the proposal No. 91X0

Electron Microscopy study: The scanning electron microscopy study was performed at UCSC Materials Analysis for Col-laborative Science (MACS) user Facility, an advanced microscopy and materials facility, located at NASA Ames Research Center in Mountain View, CA.

Dr. Ram Prasad Gandhiraman Principal Investigator, Universities Space Research Association. Mountain View, California. Email: [email protected]; [email protected]

Oct 22, 2015 Page �3






TABLE OF CONTENTS

ABSTRACT…………………………………………………………………………………… 5

INTRODUCTION……………………………………………………………………………... 6

EXPERIMENTAL WORK……………………………………………………………………. 7

CHARACTERIZATION……………………………………………………………………….13

RESULTS………………………………………………………………………………………15

CELL CULTURE……………………………………………………………………....15

SCANNING ELECTRON MICROSCOPY……………………………………………19

FOURIER TRANSFORM INFRARED SPECTRSCOPY……………………………..26

X RAY PHOTOELECTRON SPECTROSCOPY……………………………………...29

NEAR EDGE X RAY ABSORPTION FINE SPECTROSCOPY……………………...31

DISCUSSIONS…………………………………………………………………………………38

CONCLUSIONS………………………………………………………………………………..45

REFERRENCES………………………………………………………………………………..45

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Airborne Microbial Decontamination using Dielectric Barrier Discharge

Technology: Morphological and Chemical Changes

upon DBD Treatment

ABSTRACT

This study presents the morphological and chemical modification of the cell structure of

aerosolized E. coli treated with a dielectric barrier discharge (DBD). Exposure to DBD results in

severe oxidation of the bacteria, leading to the formation of hydroxyl groups and carbonyl

groups and a significant reduction in amine functionalities and phosphate groups. X-ray photoe-

mission spectroscopy (XPS) and near edge x ray absorption fine spectroscopy (NEXAFS) mea-

surements confirm the presence of additional oxide bonds upon DBD treatment suggesting oxi-

dation of the outer layer of the cell wall. Electron microscopy images show that the bacteria un-

dergo physical distortion to varying degrees from formation of pores to severe distortion of the

bacterial structure. The electromagnetic field around the DBD coil causes severe damage to the

cell structure possibly resulting in leakage of potassium ions and other vital cellular materials.

The oxidation and destruction of components of protein, DNA and lipopolysaccharides present in

the outer leaflet of the cell wall are evident from the FTIR, XPS and NEXAFS results.

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INTRODUCTION

Cold atmospheric pressure plasmas have gained significant recognition in recent years in the

field of healthcare, for example in the treatment of living cells, sterilization of medical devices,

wound healing and blood coagulation.1-3 The plasmas are nontoxic, provide rapid and continuous

anti-bacterial treatment, leave no residues to clean up and are easy to scale up, compared to other

approaches. They present an advance over radio frequency (rf) high vacuum plasmas used in the

above applications by eliminating the need for high vacuum pumps and power systems. While

the effects of atmospheric pressure plasma on surface bound bacteria have been well studied,

their use for treating airborne micro-organisms has received much less attention.4-7

The most commonly used technologies for air cleaning include high efficiency particulate air fil-

ters, UV irradiation, chemicals (spray, gel), and gas spray (ozone, hydrogen peroxide).8-13 These

methods, while popular, often provide sub-optimal outcomes such as creating bacterial resis-

tance, developing mutagenic outcomes if bacteria are under-exposed and are responsible for nu-

merous health hazards when operated incorrectly (for example, radiation exposure in the case of

UV). Prolonged exposure to radiation could cause skin irritation.14,15 UV irradiation for air disin-

fection comes in several forms, including full intensity irradiation of a room with no occupants

and shielded UV irradiation done with no restrictions on human occupation.16 One of the biggest

drawbacks of UV irradiation is that it requires direct ‘line of sight’ exposure to be effective;

‘shadowing’ may occur when used to treat airborne bacteria, a process whereby the exposed up-

per layers of a bacterial cluster are deactivated while providing a protective shield for the bacte-

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ria below them, particularly those that form biofilms.17 While the above technologies are often

used on their own, they may be combined; for example, a combination of high efficiency particu-

late air filters with UV irradiation was shown to be effective in reducing 60% of the bio-aerosols

in a contaminated room.18 Pal et. al. reported titanium dioxide nanoparticle mediated photocat-

alytic inactivation of airborne E. coli in a continuous flow reactor19,20 and fluorescent light inac-

tivation of gram negative and gram positive bacteria.21 Kowalski et.al. reported killing E. coli in

10-480 seconds using high concentrations of ozone (300-631 ppm).22

Dielectric barrier discharge (DBD) is a popular approach to produce cold plasmas at atmospheric

pressure used in air treatment.4-7In this work, we have probed the effect of DBD on the surface

chemical and topographical changes of the bacteria. Though the individual microorganisms will

have different chemical composition and cell structure, we have taken a simple gram negative

model bacteria E. coli for this study. Here, we present a DBD based technology using a novel

electrode for generating the discharge for possible air hygiene applications. E. coli was chosen as

a model organism for this study because of its prevalence in many human occupied spaces, and

its rod-like structure makes it easier to see morphological surface changes. Fourier transform in-

frared spectroscopy (FTIR) and x ray photoelectron spectroscopy (XPS) were used for the de-

tailed analysis of the chemical composition changes upon DBD exposure.

EXPERIMENTAL WORK

Materials. Escherichia coli (E. coli) (Migula) Castellani and Chalmers (ATCC 25922) was re-

hydrated in 1 ml of Tryptic soy Broth (BD 211825). The aliquot was aseptically transferred into a

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tube containing 5 ml of Tryptic Soy Broth and incubated overnight with shaking at 37 . The

bacterial suspension was then centrifuged at 1000 rpm for 10 minutes, the supernatant was dis-

carded and the cells were washed down four times with distilled water. The cell culture was

transferred to a tube and vortexed to re-suspend the bacterial cell pellet in distilled water.

Experimental Setup. The DBD system (NV200, Novaerus Inc.) electrode consists of two coax-

ial cylindrical mesh coils (304 stainless steel, 0.2 mm diameter wire) separated by a dielectric,

borosilicate glass tube (Figure 1). The glass tube has the following dimensions: 80 mm length,

22.5 mm internal diameter, and 28 mm outer diameter. A high alternating voltage, 4 kV, is ap-

plied to the coils via a step-up transformer. A fan inside the NV200 unit is used to draw the air

containing bio-aerosol into the unit. The DBD tool was placed inside a Bio-Safety Cabinet (Nu-

aire, Class II, Type A2, Model NU-425-400), and a compressor nebulizer (OMROM Compressor

nebulizer model NE-C29-E) was attached to the input of the system in order to aerosolize the

bacterial particles for testing. All vents located on the system were sealed and a platform was

placed over the top of the system to ensure the aerosolized particles had a direct route through

the system. The bacterial suspension containing E. coli and 1 ml of distilled water was trans-

ferred to the OMROM compressor nebulizer. The aerosolized particles were fed through the in-

put and any viable particles were collected at the output in Tryptic Soy Agar plates (BD 236950).

The agar plates were left in front of the output of the DBD NV200 tool and were replaced every

minute for 5 minutes. The agar plates were incubated overnight at 37 and after the incubation,

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colony-forming units (cfu) were observed on the plates as shown in Figure 2a. In these experi-

ments, 100 mm diameter petri dishes were used with both the fan and electrodes turned ON and

OFF simultaneously. In another set of experiments, the agar plates were placed continuously for

1 to 5 minutes and the cfu counts upon reculture is shown in Figure 2b. For the experiment in

Figure 2b, 60 mm diameter petri dishes were used with plasma ON and OFF, while leaving the

fan ON for both cases. The concentration of E. coli in each plate was calculated using the follow-

ing formula: bacteria/ml= number of colonies x dilution of the sample. The log reduction calcu-

lation is based on the number of viable microorganisms collected on the plate with

and without DBD.

Figure 1a

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Figure 1b

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Figure 1c

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Figure 1d

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Figure 1e

Figure 1. (a) Schematic of the NV200 DBD system, (b) diagram of the assembled coil, (c) cross

section illustrating the air flow around the dielectric barrier discharge, (d) the coil parts, and (e)

the experimental setup for inactivation E. coli and sample collection

Characterization. For scanning electron microscopy (SEM) imaging, bacterial cells on silicon

wafer were fixed in a solution of 2.5% Glutaraldehyde (Sigma-Aldrich) in Phosphate Buffered

Saline (ATCC) for 2 hours and as a second fixative procedure, in a 2% Osmium Tetroxide (Ted

Pella, Inc.) in PBS for 1.5 hours. The samples were then dehydrated in gradually increasing con-

centrations of ethanol from 60% to 100% in deionized water and chemically dried using Hexam-

ethyldisilazane (Sigma-Aldrich) for 5 minutes. They were placed in a vacuum desiccator

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overnight in order to prevent reactions with water from humidity. SEM imaging was performed

using S4800 scanning electron microscope (Hitachi, Pleasanton, CA). FTIR measurements were

carried out using a Perkin Elmer Spectrum GX system. Single side polished silicon wafers were

used as substrates for the transmission mode measurement. An untreated silicon substrate was

used as a background for the measurement and each measurement was an average of 200 scans.

The near edge x ray absorption fine spectroscopy (NEXAFS) and N1s core-level XPS measure-

ments were performed on beamline 8-2 (bending magnet endstation, spherical grating mono-

chromator) at the Stanford Synchrotron Radiation Lightsource (SSRL).23 A gold grid in the beam

path upstream of the chamber was used for the normalization of the incoming flux. The samples

were mounted on an aluminum stick with carbon tape and all the measurements were done under

UHV conditions (< 1x10-8 Torr) in a generic XPS chamber, equipped with a double pass cylin-

drical mirror analyzer (CMA, PHI 15-255G) mounted perpendicular to the incoming beam axis

in the horizontal plane. The monochromator slits and CMA pass energy (50 eV) was set for a to-

tal resolution of about 0.7eV. XPS Peak fitting was performed using Casa XPS using Shirley

background and Gaussian-Lorentzian distribution. XAS data analysis was performed using Igor

Pro. The bacterial samples collected on silicon wafer were freeze-dried using Labconco’s Free-

Zone 4.5 Liter Freeze Dry Systems.

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RESULTS

Figure 1 shows the schematic of the NV200 DBD system, diagram of the assembled coil, cross

section illustrating the airflow around the dielectric barrier discharge, the coil parts and the ex-

perimental setup for airborne inactivation of E. coli and sample collection. The bio-aerosol con-

taining the nebulized E. coli was directed at the top of the DBD tool, which has an internal fan

that pulls the contaminated air towards the DBD. The decontaminated air passes out of the bot-

tom outlet. The deactivated bacteria were collected near the outlet in a silicon wafer and agar

plates for characterization and re-culturing respectively. The purpose of a partial shield between

the nebulizer and the bottom outlet in the DBD tool is to prevent direct settlement of bacteria on

the silicon wafer and the culture plate. The DBD design shown in Figure 1 (right) contains metal

electrodes separated by a dielectric.

Figure 2a shows the reduction counts of nebulized E. coli exposed to DBD; this was collected,

re-cultured and counted. Comparison of the E. coli counts with samples collected in DBD OFF

state shows a reduction of air borne E. coli exposed to DBD. Figure 2b shows the log reduction

plot. Figure 3 shows a photograph of captured E. coli in DBD OFF and DBD ON states display-

ing the deactivation of air borne E. coli.

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Figure 2a

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Figure 2b

Figure 2. (a) E. coli counts captured from air in an agar plate and re-cultured. (a) both DBD

and fan ON, control experiment with both DBD and fan OFF in 100 mm diameter agar plate, (b)

Log reductionof aerosolized E. coli with both DBD and fan ON, control experiment with both

DBD and fan OFF in 100 mm diameter agar plate

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Figure 3. Photograph of the E. coli captured from air and re-cultured in a 100 mm diameter

petri dish containing tryptic soy agar: a) control experiment with DBD OFF and (b) DBD ON

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Scanning Electron Microscopic Images of Untreated E. coli

Figure 4a

Figure 4b

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Figure 4c

Figure 4d

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Scanning Electron Microscopic Images of DBD treated E. coli

Figure 4e

Figure 4f

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Figure 4g

Figure 4h

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Figure 4i

Figure 4j

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Figure 4k

Figure 4l

Figure 4. SEM image of (a,b) untreated E. coli drop casted from broth, (c,d) untreated E.coli

and (e-l) aerosolized E. coli passed through the DBD and captured from air

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Figure 4 shows the SEM images of the E. coli before DBD treatment (Figure 4a, 4b, 4c and 4d)

and after DBD treatment (Fig. 4e-l). The nebulized E. coli was exposed to DBD and collected

from air on a silicon wafer. Upon fixing of the microbes using the procedure described in the ex-

perimental section earlier, SEM imaging of several sets of samples was done to study the effect

of DBD on the airborne bacteria. Untreated E. coli is smooth with the surface intact and the

DBD-treated bacteria is rough with holes. The DBD alters the topographical features of E. coli to

a significant extent ranging from formation of big holes to severe structural deformation result-

ing in cell death. Physical distortion to the bacterial cell wall happens to varying degrees. It is

hypothesized that the degree of distortion depends on the proximity of the bacteria to the electric

field and the plasma discharge source. Formation of holes and structural deformation of steril-

ized E. coli similar to our observation has been reported by others using techniques such as bio-

cide emusions24 and photocatalysis.25

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Figure 5. FTIR spectroscopy analysis of untreated and DBD-treated E. coli with peak attribu-

tion. 1: free OH functionalities formed due to oxidation of hydrocarbons, 2 vibrations of amine

functionalities (amine and amide) overlapped with hydrogen bonded OH functionality, 3: alkyl

CH2 and CH3 vibrations, 4: Isocyanate or C=N functionality, 5: ether functionality of peptido-

glycan layer, 6:acid anhydride formed due to DBD treatment, 7: vibrations of amine functionali-

ties (amine and amide), 8,9: CH vibration, 10: phosphate groups present in cell membrane, 11:

P=O, C-O-C, C-O-P, and 12: OH functionality.

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The FTIR data for the untreated and DBD-treated E. coli are shown in Figure 5. The peak attri-

butions are made based on the vast FTIR spectroscopy literature on intact and damaged bacterial

cells.26-31The broad asymmetric absorption peak between 3000 and 3500 cm-1 comprises of mul-

tiple bands. Though the peaks were not distinctly resolved, the asymmetric peak shape indicates

multiple components including amides and hydrogen bonded OH groups. The characteristic

peaks at 3305 and 3066 cm−1 are assigned to amide I and amide II, respectively and the hydrogen

bonded OH groups are probably overlapped. The sharp peak centered around 3670 cm-1 in the

DBD-treated E.coli corresponds to the O-H stretching vibration of free OH groups.32 The sharp

peak centered around 2360 cm-1 in the DBD-treated E. coli is highly likely to be related to iso-

cyanate or carbon double bonded to nitrogen(C=N) bonding.32

The peak centered around 1990 cm-1 corresponds to the ether functionality of the peptidoglycan

layer. This vibration is also present in the untreated E. coli. However, a strong new peak ob-

served around 1850 cm-1 corresponds to the acid anhydride.33,34 The higher wave number 1850

cm-1 vibration corresponds to symmetric C=O stretching vibration of the anhydride and the

asymmetric stretching vibration is around 1804 cm-1 appearing as a shoulder.

A shoulder peak observed around 1730 cm-1 corresponds to C=O stretching vibrations of ester

functional groups probably from the lipids and fatty acids. The vibration bands centered around

1653 cm-1 and 1550 cm-1 correspond to amide vibrations associated with proteins. The amide I

band centered around 1653 cm-1 can be resolved into three separate peaks at 1636 cm-1, 1655 cm-

1 and 1687 cm-1. The 1655 cm-1 peak is attributed to C=O stretching vibration of amides in the α

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helical content of the protein; the peak at 1636 cm-1 corresponds to β sheets and finally, 1687 cm-

1 is related to antiparallel β sheets and aggregated strands.35-37 The amide II band centered around

1550 cm-1 band is attributed to N-H deformation of amides associated with proteins including

contributions from the C-N stretching vibrations of the peptide group. The absorption peaks be-

tween 2800 and 3000 cm-1 correspond to the stretching vibrations of alkyl groups (CH2 and CH3)

present in the bacterial cell wall fatty acids. The peaks at 2960 cm-1 and 2923 cm-1 correspond to

asymmetric stretching vibration of CH3 and CH2 and 2870 cm-1 and 2851 cm-1 correspond to

symmetric stretching vibration of CH3 and CH2 respectively. The peaks at 1460 and 1394 cm-1

correspond to CH3 and CH2 asymmetric and symmetric deformation of proteins. The presence of

a band at 1402 cm-1 has been attributed to the C-O stretching vibration of carboxylic groups.38

The peak at 1240 cm-1 is due to the P=O asymmetric stretching mode of the phospholipid phos-

phodiester backbone of nucleic acids DNA and RNA.39 The region between 1000 and 1100 cm-1

with peaks centered around 1040 and 1100 cm-1 has multiple components corresponding to the

C-O-C and C-O-P stretching of diverse polysaccharides groups and P=O symmetric stretching

overlapping with the vibrations of the sugar rings of lipopolysaccharides. The peak at 1033 cm-1

is attributed to C-OH of phosphorylated proteins and associated alcohols. The carbonyl C=O

stretching vibrations of esters and carboxylic groups from lipids and fatty acids, in general, ap-

pear around 1740 cm-1. Appearance of a new band around 930 cm-1 in the DBD treated E. coli is

due to the formation of OH species caused by oxidation. An adjacent peak around 956 cm-1 is

attributed to asymmetric stretching vibration of O-P-O functionalities.

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Figure 6a

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Figure 6b

Figure 6. N 1s Core level x ray photoemission spectrum (XPS) of (a) untreated E. coli and (b)

inactivated E. coli

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Figure 6 shows the N1s core-level XPS scan of untreated and DBD-treated E. coli. The N1s

peak of untreated E. coli can be resolved into two components and that of DBD-treated E. coli

can be resolved into three components. The major peak at 399.8 eV is due to non-protonated ni-

trogen as in amines and amides. The low intensity peak at 401.4 eV is due to protonated amines,

which are likely from the basic amino acid sequence. For the DBD-treated E. coli, an additional

peak at 402.8 eV corresponding to oxidized species is prominent.37,40,41

Figure 7a

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Figure 7b

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Figure 7c

Figure 7. a) Carbon b) Oxygen and c) Nitrogen K-Edge NEXAFS Spectra of (red) untreated and

(blue and black) DBD-treated E.coli

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The core level K-edge NEXAFS spectra of carbon, nitrogen and oxygen are displayed in Figure

7 for the untreated E. coli as well as for two DBD-treated E.coli samples with different degrees

of damage. The sensitivity of NEXAFS to the local chemical environment and the building block

character of the spectroscopy allows us to evaluate the main functional groups and their chemical

sensitivity to the DBD treatment.

Apart from its elemental specificity, XAS is particularly sensitive to the local electronic structure

including angular anisotropy in molecular orientation, bond length, oxidation state, symmetry as

well as spin. When tuning an x-ray source (synchrotron) to match a core level ionization poten-

tial, absorption of a photon can transition a core level electron into unoccupied bound or contin-

uum states. In particular, before the ionization potential, excitation to low-lying unoccupied mol-

ecular orbitals occurs, which are typically π* antibonding orbitals and mixed Rydberg/valence

states. Transition to σ* antibonding orbitals are seen above the ionization edge threshold at high-

er photon energies. XAS displays similar element specificity and local character as XPS (partici-

pation of core-hole), but with increased sensitivity to chemical environment through the unoccu-

pied states.

It is important to note that due to the presence of carbon and oxygen contaminants both from the

ambient exposure and natively at the silicon substrate surface, the absolute spectral contribution

of the E.coli relative to the substrate and absorbed contaminants is not very well defined for car-

bon and oxygen, whereas the differences between the untreated and DBD-treated samples is a

robust experimental observation.

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In the carbon spectra, we observe a low energy peak at 285.2 eV in all the samples that corre-

sponds to π* C=C transitions.42 We also observe a lower energy shoulder at 284.6 eV that might

result from normalization artifacts since it coincides with the carbon “dip” from the beamline

optics. A shoulder around 287.4 eV in all the samples reflects C-H resonances of aliphatic car-

bon. The sharp and high intensity peak at 288.4 eV in untreated E.coli can be assigned to π*C=O

transitions associated with either carboxylic groups (COOH) or the carbonyl core in CONH2.43

The shift by 0.2 eV in DBD-treated E.coli indicates an increased electronegativity in the chemi-

cal environment of the carbonyl core. A low intensity peak seen clearly at 289.4 eV in untreated

E.coli could be attributed to multiple functional groups that have transitions in this region, e.g.

C-H and O-alkyl C groups in polysaccharides or carboxylic acids, or CNH σ*.44,45The broad

peaks observed around 291 eV and 295 eV in all the samples are associated with multiple σ*

components including C-N in amino acids (~291 eV)46 and C-O in alcohols and carboxylic acits

(~292.5 eV),47,48 A low intensity peak observed around 297 eV in DBD-treated E.coli probably

corresponds to σ*C-OH. A broad excitation centered around 304 eV corresponds to C=C σ*

bonds.

The O1s NEXAFS spectra display a low energy peak near 531.6 eV for all samples that is asso-

ciated with the π* of oxygen double bond functionalities. Based on the energies of 1s->pi* in

carbonyl core groups,43 we can associate this intensity with carboxyl and CONH functionalities

(see for example, Gordon et. al., on the O XAS of fibrinogen).44 A broad peak observed between

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536 and 541 eV in untreated E.coli could be attributed to multiple components including Ryd-

berg transitions, σ*O-H and σ*C-O transitions.42,44,47 In the DBD-treated E.coli these peaks are

rather prominent around 537.3 eV and 539.5 eV, which allow us to assign them primarily to

σ*O-H and σ*C-O transitions based on the work of Ishii and Hitchcock on carboxylic acids 49

and Diaz et. al., on oxidized regions on amorphous carbon.50

The N K edge NEXAFS spectra Figure 7c shows distinct features. As Stohr et al.51-53 have

shown, there is a near linear relationship between σ* transition energies and bond lengths to

atoms adjacent to the core excited atom, the so called “bond-length with a ruler”; for example,

the shorter length of the partially conjugated amide C-N bond gives rise to a shift to higher tran-

sition energy for σ* CONH compared with σ* C-NH254 . This can be used to associate the N1s ->

σ* resonances in the NEXAFS spectrum with the main functional groups.

Low intensity peaks in the region of 388.6 to 389.8 eV correspond to N1s to π*(N=C) transi-

tions.55A high intensity peak at 401 eV corresponds to amide π* CONH transition. The peak at

405 eV in untreated E.coli could be due to multiple transitions including π* of nitro compounds,

σ* N-C and Rydberg transitions.44,56 However, for the DBD-treated E.coli, it can be concluded

from the appearance of the peak that it is highly likely to be a π* transition of nitro compounds.

This is in agreement with the literature on nitro compounds.56,57 The prominent peak centered

around 412 eV corresponds to σ*N=C as observed by Shard et. al.,58 The high energy peak

around 416 eV which is not present in untreated E.coli is likely to be σ*N=O transition.

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Rydberg Transitions

Transitions of the core electrons to another bound state of higher principal quantum number

(Rydberg orbital), which are diffuse and extended in space has been well established59,60 Excita-

tions to σ* generally will be higher in energy than those to Rydberg orbitals because the Rydberg

orbitals are nonbonding whereas the σ* MOs are antibonding and are mainly found above the

ionization potential. Stohr et.al. reported the identification of C-H resonances using NEXAFS

and its significance in the study of hydrocarbon molecules613. Outka et. al.62 observed strong C-H

resonances for condensed alcohols on Si surfaces at 288.1 eV and 289.4 eV and C-C σ* reso-

nance 292.8 eV. Knorr et.al. in their NEXAFS study of amino silane assigned 289 and 290 eV

peaks to C-H resonances63.

In the presence of bonds to hydrogen atoms, mixing of Rydberg orbitals with hydrogen-derived

antibonding orbitals of the same symmetry increases the intensity of the corresponding reso-

nance64,65. Urquhart et al. in their study on gaseous alkanes observed that the valence character

decreases as the number of C-H bonds decreases and concluded that in gaseous alkanes there can

only be Rydberg valence mixing when there is a valence orbital of appropriate symmetry and

energy64. Urquhart et.al.66 in their study on gaseous and condensed phase neopentane observed

that both the theoretical and experimental results showed quenching of Rydberg states in solid

phase. Weiss et al.67 studied in detail the Rydberg transitions in condensed alkanes located be-

tween 287.4 and 288.1 eV and reported that the excited Rydberg states are not quenched but re-

tain a definite excitonic character and are blue shifted. Strong presence of Rydberg states in

Oct 22, 2015 Page �37


solids is demonstrated by Bagus et al.68 and they also reported that the C K edge NEXAFS spec-

tra of alkanes show pronounced features below the ionization potential that are best described as

mixed Rydberg/Valence resonances. Ishi and Hitchcock69 in their investigation of alcohols at-

tributed the 289.4/289.2 eV peaks to excitations of a mixed Rydberg/Valence orbital.

Urquhart et.al.70 reported N1s ->3s and N1s ->3p Rydberg transitions in gas phase urea at 401.5

eV and 402.7 eV respectively, indicating a decrease in Rydberg transitions with decreasing NH2

groups. In a comparison of gaseous and condensed glycine, Gordon et.al.71observed a striking

difference between the gaseous and condensed phase Rydberg transitions. For gaseous glycine,

the peaks at 401.3 eV and 402.5 eV were assigned as N1s->3S Rydberg and N1s->3p Rydberg

transition respectively. However, both these features were absent in the condensed phase. Otero

et.al.72 studied the N1s NEXAFS of amino acids and concluded that the attenuation of Rydberg

transitions in condensed phase is due to intermolecular effect, where the presence of neighboring

molecules in the solid disrupts the Rydberg ortibals leading to their attenuation.

DISCUSSION

DBD-based inactivation of the aerosolized E. coli as observed in Figures 2 and 3 can be caused

by multiple factors. The gram negative bacteria contain cytoplasmic membrane, peptidoglycan

layer and outer membrane. The ability of the cell to withstand high internal osmotic pressure and

the maintenance of the cell shape is due to the peptidoglycan layer that consists of amino sugars

and amino acids.73,74 The outer membrane contains structural proteins, receptor molecules and

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phospholipids and acts as a selective permeability barrier containing hydrophilic diffusion chan-

nels.75-77 It has been reported that the change in permeability of the membrane during the inacti-

vation process results in leakage of potassium ion (K+), a vital component involved in protein

synthesis and essential for cell viability.78,79 The formation of pores, as observed in the SEM im-

ages, is more likely to have caused potassium ion leak resulting in cell death. The structural de-

formation suggests extensive chemical degradation of the microbe through the action of radicals

and/or charged species generated by DBD.

The role of hydroxyl radicals and reactive oxygen species in E.coli inactivation has been studied

by several research groups.80,81The extent of this molecular damage was assessed using infrared

spectroscopy by observing the major chemical components such as C=O and N-H vibrations of

amides associated with proteins, C=O vibrations of esters from lipids, CH vibrations of the bac-

terial cell walls, P=O vibrations of the phosphodiester of nucleic acids and the C-O-C vibrations

of polysaccharides.26 The formation of free and hydrogen bonded hydroxyl groups in the decon-

taminated E. coli has been observed here.

Exposure of the nebulized bacteria in air to DBD resulted in a significant change in chemical

structure. Significant reduction in the intensity of broad stretching vibrations of amine between

3000 to 3500 cm-1 and the formation of hydrogen bonded and free hydroxyl groups between

3500 to 4000 cm-1 is very evident. An intense peak centered around 3665 cm-1 corresponds to

free OH functionalities. The hydrogen bonded OH as seen in untreated E. coli between 3000 to

3500 cm-1 is broad and the sharpness of this peak shifting to higher wave number in the DBD-

treated E. coli confirms that the OH groups are not hydrogen bonded but are free. A strong new

Oct 22, 2015 Page �39


peak observed around 1850 cm-1 in the DBD-treated E. coli corresponds to the acid anhydride.

The absence of this peak in the untreated E. coli and the strong vibration observed upon DBD

treatment is an indication that this chemical functionality is a product of interaction of the E. coli

with reactive species generated by the DBD.

The C=O stretching vibration and N-H deformation of amides associated with the proteins in the

range of 1655 and 1550 cm-1 show a very clear variation. The decreased intensity of the amine

and amide vibrations could probably be due to the formation of C=N groups, as observed by the

new peak at 2360 cm-1, by replacement of the carbonyl oxygen in amide with nitrogen, probably

from the primary amine. The reaction mechanism is unpredictable as it is a complex structure;

however, the presence of strong vibration at this wavenumber and the reduced intensity of the

amide and primary amine vibrations around 1650 and 1550 cm-1 can be correlated with this ob-

servation. An enormous increase in the intensity of the peak centered around 1100 cm-1 could be

attributed to the oxidation of P=O, C-O-C and C-O-P groups of lipopolysaccharides and other

structural proteins and receptor molecules present in the outer layer. In the DBD-treated E. coli,

the appearance of a new band caused by oxidation has been observed at around 930 cm-1 due to

the OH species and an adjacent peak around 956 cm-1 attributed to asymmetric stretching vibra-

tion of O-P-O functionalities. All these observations suggest damage to the outer leaflet of the E.

coli due to the exposure to the DBD. The presence of these two strong vibrational modes at 3650

cm-1 and 1850 cm-1 along with a sharp peak at 1100 cm-1, on the DBD-treated E. coli is a strong

indication of the role of the DBD-generated reactive oxygen species in causing the chemical

changes possibly resulting in cell death.

Oct 22, 2015 Page �40


In both the untreated and DBD-treated E. coli, the N1s core-level photoemission spectra con-

firms the presence of non-protonated nitrogen as in amines and amides and protonated amines

which are likely from the basic amino acid sequence. For the DBD-treated E. coli, the observa-

tion of oxidized species (additional peak at 402.8 eV) suggests oxidation of the outer layer of the

cell wall (the mean probing depth in these XPS measurements is about 2-3 nm).

The major difference between untreated E.coli and DBD-treated E.coli arises from the carbon-

oxygen bonding environments including carbonyl core transitions of amide and carboxylic func-

tionalities. In the DBD-treated E.coli, the shift of π* CONH carbonyl core peak by 0.2 eV (from

288.4 eV to 288.6 eV) suggests the oxidation of the amide group as the electronegative group

will shift the peak to higher energy. This peak attribution is based on the trends in carbonyl core

(C=O) π* transitions, as a function of electronegativity of the chemical environment, as reported

by Urquhart and Ade.43 Gordon et. al., in their study on inner shell excitation spectroscopy of the

peptide bonds and proteins, report that the carbonyl core π* (C=O) transition occurs between

288.2 eV to 288.6 eV.44 Here the 289.4 eV peak corresponding to π* of O-alkyl C group in poly-

saccharides or carboxylic acids disappeared or decreased in intensity upon DBD treatment sug-

gesting changes either in the polysaccharide network or in carboxyl functionality of the cell wall.

The relative intensities of the π* carbonyl core transition at 288.4 eV and C-O σ* transition at

292.5 eV show a very clear difference between untreated and DBD-treated E.coli. The signal in-

tensity corresponding to C-O σ* (292.5 eV) is relatively higher in DBD-treated E. coli resulting

in decreased π*C=O / σ*C-O ratio. The increase in C-O σ* in DBD sample can be correlated to

Oct 22, 2015 Page �41


the decreased intensity of the carbonyl core of amide (288.6 eV) and that of carboxylic groups

(289.4 eV).

O XAS shows very interesting features, which agree with C XAS and FTIR observations. O1s to

π* C=O transition is observed at 531.6 eV for all the samples. However, the intensity of this peak

is diminished to a significant extent in DBD-treated E.coli. Broad feature between 537 to 540 eV

in the untreated E.coli can be associated with ether groups (-C-O-C-), and alcohols (-OH) based

on comparison with condensed alcohols as well as liquids, for which some degree of hydrogen

bonding reduces (-OH) resonance clearly. Pylkkanen et. al., in their study on the signature of hy-

drogen bonding in the oxygen K edge spectrum of alcohols report that hydrogen bonding results

in dampening of the pre-edge and broadening of the main edge.82 Well-defined (-OH) that are

separated (as could be expected from repulsion) would give rise to a more distinct peak before

the main edge. For DBD-treated E.coli, the peaks at 537.3 and 539.5 eV, corresponding to σ*O-

H and σ*C-O transitions respectively, are prominent with increased intensity indicating the oxi-

dation of the surface and formation of free OH groups in agreement with the FTIR. Also, the ra-

tio of π*C=O (531.5 eV) to σ*OH (537.3 eV) or σ*C-O(539.5eV) shows a trend very similar to

that of the C XAS. An increased σ* O-H and σ* C-O intensity and decreased π* C=O intensity

correlates with the C XAS data. It should also be noted that the decreased intensity of π* CONH

transition in DBD-treated E.coli is in high correlation with the FTIR spectrum where the vibra-

tions corresponding to amide (1650 and 1550 cm-1) decreased in intensity upon DBD treatment.

It is evident from C XAS and O XAS that the π*C=O transitions are reduced in intensity after

DBD treatment i.e., the unsaturated bonds in carbon are decreased. The DBD-generated reactive

Oct 22, 2015 Page �42


species oxidize both alkyl and carbonyl groups. FTIR, C1s XPS and O XAS confirm the forma-

tion of strong saturated functionalities resulting in varying local configurations. O XAS shows

formation of single bonded oxygen bonds including OH and CO bonds as evident from strong

peaks at 537.3 and 539.5 eV.

N 1s XAS spectra show an apparent difference between untreated and DBD-treated E.coli. The

peak at 401 eV corresponding to amide π* CONH transition shows a clear difference between

upon DBD treatment. The decrease in 401 eV peak intensity upon DBD treatment is in correla-

tion with the C XAS (288.6 eV) and O XAS (531.5 eV) and FTIR(1550- 1700 cm-1). A sharp

rise in 405 eV peak corresponding to π* transition of nitro group is a clear evidence of oxidation

of the cell surface resulting in drastic chemical changes. The transition corresponding to σ*N=C

is high intensity and prominent in samples treated by DBD. This is also in agreement with the

FTIR spectra where the sharp peak centered around 2360 cm-1 in the DBD-treated E. coli, corre-

sponding to C=N bonding vibration, is of high intensity and prominent upon DBD treatment.

The inactivation mechanism of the reactive oxygen species is based on its effect on cell mem-

brane and intra cellular substances. As hydroxyl groups are permeable to cell membrane, the hy-

droxyl species penetrate the E. coli cell membrane, react with cytoplasmic substances and cause

chromosomal DNA damage resulting in the deactivation.83 This effect is clearly observed in Fig-

ure 5 with the disappearance of one of the amide related peaks and an intense carbonyl peak

formation.

Airborne microbial load in closed environments including office space and hospitals vary signif-

icantly across the world. Reports on air quality studies carried out in hospitals vary from 5 to 25

Oct 22, 2015 Page �43


cfu/m3 in certain countries to more than 2500 cfu/m3 in some other countries depending on the

personnel density, climate conditions etc.84-87 The log reduction reported here is greater than log

0.5 within 5 minutes of operation. This corresponds to the inactivation of more than 68% of the

microbes passed through the DBD system. While this percentage is lower than kill rates quoted

by various surface cleaning disinfectants (e.g. 99 % or higher), it should be noted that the micro-

bial load for the tests reported here exceeded 10,000,000 cfu/m3 while the log reduction calcula-

tion is based on the number of viable microbes settled on the plate under DBD on and DBD off

conditions. Also, the ability of this system to inactivate very low concentration of pathogenic

bacteria in real air samples needs to be studied. Though the present initial study demonstrates

that the DBD can inactivate the aerosolized E. coli, the type of bacteria we have used and the re-

sults presented here alone are not sufficient to claim the ability of this system to provide safe en-

vironment to hospitals or work places. We have used non-pathogenic biosafety level 1 E. coli,

but the actual environment in hospitals contains spores, molds and other robust gram positive

bacteria in addition to E. coli. Further inactivation studies of other types of pathogenic bacteria

need to be done to determine the efficacy of the system and the discussion on other pathogens is

beyond the scope of this paper.

Oct 22, 2015 Page �44


CONCLUSIONS

The effect of dielectric barrier discharge on the morphology and surface chemistry of E. coli was

studied using electron microscopy, infrared spectroscopy and x ray absorption spectroscopy. The

electromagnetic field around the DBD coil causes severe distortion of the morphology of E. coli

to varying degrees from formation of pores to shrinking and elongation of the cell structure pos-

sibly resulting in leakage of potassium ion. The observations made from the core level K-edge

NEXAFS spectra of carbon, nitrogen and oxygen correlate well with each other as well as with

the FTIR spectra concluding the chemical modification of the cell structure. The oxidation and

destruction of components of protein, DNA and lipopolysaccharides present in the outer leaflet

of the cell wall are evident from FTIR, XPS and NEXAFS spectroscopy.

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