Development of copper based material systems forgenerating nitric oxide to control nitrifying bacterial biofilms

Author:Wonoputri, Vita

Publication Date:2016

DOI:https://doi.org/10.26190/unsworks/19322

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Development of Copper Based Material

Systems for Generating Nitric Oxide to

Control Nitrifying Bacterial Biofilms

A thesis submitted to The University of New South Wales in

partial fulfilment of the degree of Doctor of Philosophy

By

Vita Wonoputri

School of Chemical Engineering

Faculty of Engineering

December 2016

i

THE UNIVERSITY OF NEW SOUTH WALES

Thesis/Dissertation Sheet Surname or Family name: WONOPUTRI

First name: VITA

Other name/s:

Abbreviation for degree as given in the University calendar:

PhD

School: Chemical Engineering

Faculty: Engineering

Title: Development of Copper Based Material Systems for Generating Nitric Oxide to Control Nitrifying Bacterial Biofilms

Abstract 350 words maximum: (PLEASE TYPE) Biofilms, which are bacteria cells aggregates that exist within a matrix of extracellular polymeric substances, are

known to cause problems in industrial water systems. In order to prevent biofilm formation and pathogens occurrence, disinfectants such as chloramine are usually added into the water. However, chloramine addition has been shown to trigger the growth of nitrifying biofilms which subsequently accelerate chloramine decay. Therefore, a new antibiofilm agent, namely nitric oxide (NO), is investigated for nitrifying biofilm control.

Herein, NO was generated from catalytic reduction of nitrite in the presence of a copper(II) complex catalyst embedded in a poly(vinyl chloride) (PVC) matrix. Ascorbic acid was added into the solution as a reducing agent to aid the formation of the active copper(I) species that will react with nitrite to generate NO. The copper-nitrite-ascorbic acid combination showed enhanced NO generation compared to that generated in the presence of nitrite-ascorbic acid alone, and subsequently, enhanced biofilm suppression was observed. The catalytically generated NO was also found to be effective in dispersing pre-formed biofilm, with simultaneous biofilm cells killing observed when a high concentration of nitrite-ascorbic acid was used.

An alternative reducing agent, namely Fe2+, was investigated for the potential to mediate reduction of copper(II) complex for generating NO. The amount of NO generated was found to be highly dependent on Fe speciation in different pH and buffer composition. Nonetheless, the NO generated in phosphate buffer pH 6 is still capable of dispersing pre-formed nitrifying biofilms, thus suggesting the robustness of NO-mediated biofilm dispersal.

In the last part of the study, an iron complex, namely FeDTTCT, was synthesised and immobilised into PVC with the copper catalyst. The iron complex was found to facilitate copper(II)/copper(I) redox cycling, subsequently enabling NO generation from nitrite. Importantly, the mixed metal system exhibited a non-toxic antibiofilm activity, whereby biofilm formation was suppressed and bacterial growth was confined to the free-floating planktonic phase. These thus imply that the mixed metal system was capable of converting nitrite endogenously produced by nitrifying bacteria to NO, hence eliminating the need to add a reducing agent and NO precursor in solution form into the system.

Declaration relating to disposition of project thesis/dissertation I hereby grant to the University of New South Wales or its agents the right to archive and to make available my thesis or dissertation in whole or in part in the University libraries in all forms of media, now or here after known, subject to the provisions of the Copyright Act 1968. I retain all property rights, such as patent rights. I also retain the right to use in future works (such as articles or books) all or part of this thesis or dissertation. I also authorise University Microfilms to use the 350 word abstract of my thesis in Dissertation Abstracts International (this is applicable to doctoral theses only). ………………………………………… Signature

……………………………………..…………….. Witness Signature

……….…………………….… Date

FOR OFFICE USE ONLY

Date of completion of requirements for Award:

ii

ORIGINALITY STATEMENT

‘I hereby declare that this submission is my own work and to the best of my knowledge it contains no materials previously published or written by another person, or substantial proportions of material which have been accepted for the award of any other degree or diploma at UNSW or any other educational institution, except where due acknowledgement is made in the thesis. Any contribution made to the research by others, with whom I have worked at UNSW or elsewhere, is explicitly acknowledged in the thesis. I also declare that the intellectual content of this thesis is the product of my own work, except to the extent that assistance from others in the project's design and conception or in style, presentation and linguistic expression is acknowledged.’

Signed ……………………………………………..............

Date ……………………………………………..............

iii

COPYRIGHT STATEMENT

‘I hereby grant the University of New South Wales or its agents the right to archive and to make available my thesis or dissertation in whole or part in the University libraries in all forms of media, now or here after known, subject to the provisions of the Copyright Act 1968. I retain all proprietary rights, such as patent rights. I also retain the right to use in future works (such as articles or books) all or part of this thesis or dissertation. I also authorise University Microfilms to use the 350 word abstract of my thesis in Dissertation Abstract International (this is applicable to doctoral theses only). I have either used no substantial portions of copyright material in my thesis or I have obtained permission to use copyright material; where permission has not been granted I have applied/will apply for a partial restriction of the digital copy of my thesis or dissertation.' Signed ……………………………………………........................... Date ……………………………………………...........................

AUTHENTICITY STATEMENT ‘I certify that the Library deposit digital copy is a direct equivalent of the final officially approved version of my thesis. No emendation of content has occurred and if there are any minor variations in formatting, they are the result of the conversion to digital format.’ Signed ……………………………………………...........................

Date ……………………………………………...........................

Abstract

iv

Abstract

Biofilms, which are bacteria cells aggregates that exist within a matrix of

extracellular polymeric substances, are known to cause problems in industrial water

systems. In order to prevent biofilm formation and pathogens occurrence, disinfectants

such as chloramine are usually added into the water. However, chloramine addition has

been shown to trigger the growth of nitrifying biofilms which subsequently accelerate

chloramine decay. Therefore, a new antibiofilm agent, namely nitric oxide (NO), is

investigated for nitrifying biofilm control.

Herein, NO was generated from catalytic reduction of nitrite in the presence of a

copper(II) complex catalyst embedded in a poly(vinyl chloride) (PVC) matrix. Ascorbic

acid was added into the solution as a reducing agent to aid the formation of the active

copper(I) species that will react with nitrite to generate NO. The copper-nitrite-ascorbic

acid combination showed enhanced NO generation compared to that generated in the

presence of nitrite-ascorbic acid alone, and subsequently, enhanced biofilm suppression

was observed. The catalytically generated NO was also found to be effective in

dispersing pre-formed biofilm, with simultaneous biofilm cells killing observed when a

high concentration of nitrite-ascorbic acid was used.

An alternative reducing agent, namely Fe2+, was investigated for the potential to

mediate reduction of copper(II) complex for generating NO. The amount of NO

generated was found to be highly dependent on Fe speciation in different pH and buffer

composition. Nonetheless, the NO generated in phosphate buffer pH 6 is still capable of

Abstract

v

dispersing pre-formed nitrifying biofilms, thus suggesting the robustness of NO-

mediated biofilm dispersal.

In the last part of the study, an iron complex, namely FeDTTCT, was

synthesised and immobilised into PVC with the copper catalyst. The iron complex was

found to facilitate copper(II)/copper(I) redox cycling, subsequently enabling NO

generation from nitrite. Importantly, the mixed metal system exhibited a non-toxic

antibiofilm activity, whereby biofilm formation was suppressed and bacterial growth

was confined to the free-floating planktonic phase. These thus imply that the mixed

metal system was capable of converting nitrite endogenously produced by nitrifying

bacteria to NO, hence eliminating the need to add a reducing agent and NO precursor in

solution form into the system.

Acknowledgements

vi

Acknowledgements

This PhD thesis would not be possible without my supervisors. My sincere

gratitude goes to Prof. Rose Amal for giving me this opportunity and for being a

wonderful mentor. Thanks to Dr. Lachlan Yee and Dr. Nicolas Barraud for all the help

in developing this lovely project and for all the constructive feedback. My gratitude also

goes to Dr. Sanly Liu for always guiding and helping me and Dr. Cindy Gunawan for all

her valuable feedback. Thank you Dr. May Lim for all the support throughout my PhD

study. This research was supported under Australian Research Council’s Linkage

Projects funding scheme (LP110100459). The financial supports from Australian Water

Quality Centre (SA Water) and Water Corporation (Western Australia) and the PVC

provision from Chemson Pacific Pty Ltd are gratefully acknowledged.

The assistance from Mark Wainwright Analytical Centre throughout this PhD

project is much appreciated. Special thanks to Dr. Bill Gong for XPS analysis, Dr.

Rabeya Akter and Dr. Dorothy Yu for ICP and ion chromatography analysis, and all the

people in BMIF and NMR lab. My sincerest gratitude to Mr. Pejhman Keshvardoust for

sharing the knowledge about nitrifying bacteria. Thank you to Dr. Victor Wong for

always helping me with the consumables ordering and John Starling for all the lab

assistance. I also would like to extend my gratitude to Mr. Jim Paschali, Mr. Phil

Thompson, and Mr. Paul Brockbank for all the technical support, especially for fixing

my incubator. Administrative support from Ms. Ik Ling Lau is much appreciated. I give

my special thanks to Dr. Mandalena Hermawan, the queen bee of Partcat gossip, for all

the support and laughter.

Acknowledgements

vii

Thank you to my Partcat friends, especially bio-lab partner Ayu, partner-in-crime

Emma, my first friend Shi Nee, gym buddy Ee Teng, and all the girls who have kept me

well fed: Hui Ling, Cui Ying, Xue Lian and Phoebe. To the Partcat boys: Tze Hao Tan,

RJ, and Hendra, thanks for enduring all the teasing. Special thanks to Peng Wang and

Amir Nashed for all the chatting session. The supports from all past and present

members of Partcat are much appreciated. My gratitude also goes to my friends in

CMBB (616).

Thank you for all the loving support from my family back in Indonesia: my dad

who always pushes me to work to the best of my ability, my mom for always praying

for me (and for sending me cute pictures of my pomeranian Science!), and my sister and

her husband for always supporting me. Finally, to my dear husband, thanks for letting

me pursue my dream.

List of Publications

viii

List of Publications

JOURNAL PUBLICATIONS:

1. Wonoputri, V.; Gunawan, C.; Liu, S.; Barraud, N.; Yee, L. H.; Lim, M.; Amal,

R. Copper complex in poly(vinyl chloride) as a nitric oxide-generating catalyst

for the control of nitrifying bacterial biofilms. ACS Applied Materials and

Interfaces (2015), 7 (40), 22148–22156.

2. Wonoputri, V.; Gunawan, C.; Liu, S.; Barraud, N.; Yee, L. H.; Lim, M.; Amal,

R. Iron complex facilitated copper redox cycling for nitric oxide generation as

non-toxic nitrifying biofilm inhibitor. ACS Applied Materials and Interfaces

(2016), 8(44), 30502-30510.

3. Wonoputri, V.; Gunawan, C.; Liu, S.; Barraud, N.; Yee, L. H.; Lim, M.; Amal,

R. Ferrous ion as reducing agent in the generation of antibiofilm nitric oxide

from copper-based catalytic system (in preparation)

CONFERENCE PRESENTATIONS:

1. Wonoputri, V.; Liu, S.; Gunawan, C.; Barraud, N.; Yee, L. H.; Lim, M.; Amal,

R. Suppression and removal of nitrifying bacteria biofilm by catalytic

generation of nitric oxide. Australasian Particle Technology Society 2nd Student

Conference, Phillip Island, Australia, 26-27 September 2015.

2. Wonoputri, V.; Liu, S.; Gunawan, C.; Barraud, N.; Yee, L. H.; Lim, M.; Amal,

R. Efficacy and mechanism of action of nitric oxide against nitrifying bacteria

biofilms at different stages of development. Asian Pacific Confederation of

List of Publications

ix

Chemical Engineering, Melbourne, Australia, 27 September-1 October 2015

(poster).

3. Wonoputri, V.; Liu, S.; Gunawan, C.; Barraud, N.; Yee, L. H.; Lim, M.; Amal,

R. Catalytic generation of nitric oxide for the control of nitrifying bacteria

biofilm. 7th ASM Conference on Biofilms, Chicago, USA, 24-29 October 2015

(poster).

4. Wonoputri, V.; Liu, S.; Gunawan, C.; Barraud, N.; Yee, L. H.; Lim, M.; Amal,

R. Nitrifying biofilm control through the utilisation of catalytically generated

nitric oxide. The University of New South Wales Engineering Postgraduate

Research Symposium, Sydney, Australia, 9-11 November 2015.

5. Wonoputri, V.; Liu, S.; Gunawan, C.; Barraud, N.; Yee, L. H.; Lim, M.; Amal,

R. Control of nitrifying bacteria biofilms through catalytically generated nitric

oxide, invited speaker for iThree Institute group presentation, University of

Technology Sydney, 9 August 2016.

x

Table of Contents

Abstract ........................................................................................................................... iv

Acknowledgements ......................................................................................................... vi

List of Publications ....................................................................................................... viii

Table of Contents ............................................................................................................ x

Chapter 1. Introduction ............................................................................................... 1

1.1. References .......................................................................................................... 4

Chapter 2. Literature review ....................................................................................... 8

2.1. Biofilms: introduction and properties ................................................................. 8

2.2. Issues with biofilms presence and difficulties in its eradication ...................... 14

2.3. Current strategies for biofilm prevention and removal .................................... 20

2.3.1. Adhesion prevention ................................................................................. 21

2.3.2. Biofilm cells killing and biofilm maturation inhibitor .............................. 26

2.3.3. Biofilm disruption ..................................................................................... 34

2.3.4. Nitric oxide as a new antibiofilm agent .................................................... 37

2.4. Current strategies in nitric oxide utilisation ..................................................... 38

2.4.1. Nitric oxide-releasing materials for application in biomedical field ........ 38

2.4.2. Application of nitric oxide donors in water industry ................................ 45

2.5. Summary........................................................................................................... 46

2.6. References ........................................................................................................ 47

Chapter 3. Nitric oxide generation from nitrite and ascorbic acid solution in

the presence of copper complex catalyst ..................................................................... 63

3.1. Introduction ...................................................................................................... 63

3.2. Experimental methods ...................................................................................... 66

Chapter 1

xi

3.2.1. Synthesis of copper dibenzo[e,k]-2,3,8,9-tetraphenyl-1,4,7,10-tetraaza-

cyclododeca-1,3,7,9-tetraene complex (CuDTTCT complex) and its

characterisation ........................................................................................................ 66

3.2.2. NO generation measurement ..................................................................... 68

3.2.3. Ascorbic acid oxidation measurement ...................................................... 69

3.2.4. Biofilm suppression assay ......................................................................... 69

3.2.5. Biofilm dispersal and metabolic activity assay on pre-formed biofilms... 71

3.3. Results and discussion ...................................................................................... 72

3.3.1. Characterisation of copper complex (CuDTTCT) powder and films ....... 72

3.3.2. Nitric oxide generation measurements ...................................................... 74

3.3.3. Nitrifying bacteria biofilm suppression by nitrite-ascorbic acid and

CuDTTCT-nitrite-ascorbic acid mixture ................................................................. 76

3.3.4. Nitrifying bacteria biofilm dispersal upon the addition of nitrite-ascorbic

acid in the presence of CuDTTCT ........................................................................... 83

3.4. Summary........................................................................................................... 88

3.5. References ........................................................................................................ 89

Chapter 4. Catalytic generation of nitric oxide: the use of ferrous ion as a

reducing agent ............................................................................................................... 94

4.1. Introduction ...................................................................................................... 94

4.2. Experimental methods ...................................................................................... 96

4.2.1. Synthesis of copper dibenzo[e,k]-2,3,8,9-tetraphenyl-1,4,7,10-tetraaza-

cyclododeca-1,3,7,9-tetraene complex (CuDTTCT complex) ................................ 96

4.2.2. NO generation measurement with Fe2+ solution as the reducing agent .... 96

4.2.3. Fe speciation analysis ................................................................................ 97

4.2.4. Biofilm dispersal assay ............................................................................. 97

4.3. Result and discussion ....................................................................................... 98

4.3.1. Effect of testing solution and pH on NO generation from CuDTTCT-

nitrite-Fe2+ mixture .................................................................................................. 98

Chapter 1

xii

4.3.2. Effect of nitrite and Fe2+ concentration on NO generation from

CuDTTCT-nitrite-Fe2+ mixture ............................................................................. 106

4.3.3. Biofilm dispersal in the presence of CuDTTCT-nitrite-Fe2+ solution .... 109

4.4. Summary......................................................................................................... 113

4.5. References ...................................................................................................... 114

Chapter 5. Iron complex facilitated copper redox cycling for nitric oxide

generation ................................................................................................................. 117

5.1. Introduction .................................................................................................... 117

5.2. Experimental methods .................................................................................... 119

5.2.1. Synthesis of dibenzo[e,k]-2,3,8,9-tetraphenyl-1,4,7,10-tetraaza-

cyclododeca-1,3,7,9-tetraene ligand and the metal complexes ............................. 119

5.2.2. Characterisation of metal complexes ...................................................... 120

5.2.3. NO generation measurement ................................................................... 121

5.2.4. Biofilm assay ........................................................................................... 122

5.3. Result and discussion ..................................................................................... 123

5.3.1. Confirmation of metal complexes formation .......................................... 123

5.3.2. Nitric oxide generation from CuDTTCT+FeDTTCT films in the presence

of nitrite ................................................................................................................. 125

5.3.3. Antibiofilm activity of CuDTTCT+FeDTTCT mixed metal film by the

involvement of catalytically generated NO ........................................................... 130

5.4. Summary......................................................................................................... 138

5.5. References ...................................................................................................... 139

Chapter 6. Conclusions and Recommendations .................................................... 143

6.1. Conclusions .................................................................................................... 143

6.2. Recommendations .......................................................................................... 145

6.3. References ...................................................................................................... 149

1

Chapter 1.

Introduction

In their natural habitat, bacteria have a tendency to form surface-attached

communities known as biofilms, where their formation is identifiable by the production

of extracellular polymeric substances that encased and protect the bacteria.1 Biofilms

formation has detrimental effects in many areas, ranging from health care to industrial

water system. For instance, biofilms can decrease heat exchanger or cooling tower

efficiency.2 Biofilm presence can foul water filtration membranes, instigating the need

for more frequent cleaning, which subsequently reduces the membrane lifetime.3

Biofilm formation on water distribution pipelines can lead to microbial induced

corrosion and offer protection for pathogenic bacteria, potentially releasing them into

the flowing water through the natural shedding cycle of biofilm or shear force.4

Moreover, environmental biofilms have been found to act as a reservoir for the spread

of antibiotics resistance genes, due to the high cell density and close cell-to-cell

proximity.5,6

In order to control microbial growth, disinfectants such as chlorine and

chloramine are usually added into the water. However, these disinfectants have been

shown to be ineffective at eradicating biofilms, due to the presence of protective EPS

barrier which increased biofilm cells resistance to antimicrobial compounds compared

to their free-floating (planktonic) counterparts.7 Chloramine disinfectant, which is

Chapter 1

2

preferred compared to chlorine because of less formation of disinfection by-products

and more stable compared to chlorine, is proven to be ineffective towards the ubiquitous

environmental biofilm of nitrifying bacteria.8,9 Moreover, nitrifying bacteria and their

soluble microbial products can accelerate the inactivation and decay of chloramine,

subsequently releasing chlorine and ammonia, where the latter act as the main nutrient

for nitrifying bacteria, further enhancing the biofilm formation.10–12 Therefore, there is a

clear need to develop a novel method to eradicate biofilms with minimal potential for

bacterial resistance development.

Several novel antibiofilm strategies have been proposed, which generally can be

divided into three different methods depending on the mechanism employed: adhesion

prevention, biofilm cells killing, and disruption of biofilm.13 However, it is believed that

a single method would not be able to eradicate biofilm thoroughly. Therefore, a

combination of different mechanisms is preferred. Nitric oxide (NO), which is a soluble

free radical gas that has been shown to be effective at controlling the formation of

antimicrobial-resistant biofilms,14 seems to be an attractive solution. NO can affect

biofilms either by inducing a toxic pathway (cell death) at high concentrations or via a

non-toxic pathway at low concentrations that cause biofilm bacteria to disperse from the

surface and revert to a free-floating phase.15–17 Importantly, it is believed that biofilm

treatment by NO will not trigger the development of resistant biofilms, due to the

several mechanisms by which NO presents toxicity to cells15,18 or the signalling

pathway involved in its non-toxic antibiofilm effect.19 However, application of NO is

still limited due to its high reactivity and short half-life. These properties of NO have

led to the development of novel strategies to control biofilms, in particular on materials

that are capable of delivering NO to the target site.

Chapter 1

3

In general, materials that are capable of delivering NO can be divided into two

types: NO-releasing materials and NO-generating materials.20–22 NO-releasing materials

depend on the use of NO donors such as N-diazeniumdiolates (NONOates) or S-

nitrosothiols (RSNO) which are incorporated into a nanomaterial delivery vehicle or

coating. Such materials have limited NO release due to the finite reservoir of NO that

can be loaded during synthesis. In contrast, NO-generating materials appear to be more

advantageous due to their ability to catalyse NO generation from endogenous sources,

such as nitrite ions which are ubiquitous in water.23 However, the ability of NO-

generating materials in eradicating biofilms has yet to be determined. Moreover, such

materials, which are usually copper(II) or selenium(II) based, are highly dependent on

the presence of appropriate reducing agents, as the reduced form is the most active

species in catalytic generation of NO.21,24 Therefore, this thesis attempts to provide

insight on the utilisation and development of copper-based NO-generating materials for

controlling the formation of biofilms. Nitrifying bacteria, commonly occurring in

chloraminated industrial water system, were chosen as a biofilm-forming model

organism. In specific, the aims and objectives of the work presented in this thesis are:

1. To assess the ability of catalytic generation of NO for the prevention and

removal of nitrifying bacteria biofilms.

2. To examine the use of Fe2+ ions as an alternative reducing agent for instigating

copper redox cycling for NO generation and its subsequent effect on biofilm

removal.

3. To develop an iron-based complex that can be embedded together with the

catalyst to facilitate copper redox cycling for generating NO, therefore

eliminating the need to add any reducing agent in the water.

Chapter 1

4

Chapter 2 provides background on biofilm properties and implications, and

reviewed the current literature on biofilm control strategies, with a detailed focus on

NO-based control strategy and materials. Chapter 3 presents an investigation on the use

of a copper complex catalyst immobilised within a poly(vinyl chloride) (PVC) matrix to

generate NO in the presence of nitrite as an NO source, and ascorbic acid as a reducing

agent. The effectivity of the system to prevent and remove nitrifying bacteria biofilm

was also investigated. Chapter 4 describes a comprehensive study on the use of Fe2+

ions as an alternative reducing agent for catalytic NO generation, including

determination of iron speciation, investigations of the effect of different parameters,

such as pH, solution composition, and nitrite-Fe2+ concentration toward NO production,

and the resulting biofilm removal efficiency. Following the result presented in Chapter

4, an iron complex that can facilitate copper redox cycling for NO generation was

synthesised and results are detailed in Chapter 5. The iron complex was subsequently

embedded together with the copper catalyst within a PVC matrix, eliminating the need

for the addition of reducing agent in solution form. PVC films containing the mixed

metal complexes were then examined for biofilm formation in the presence of nitrite

endogenously produced by the nitrifying bacteria. Chapter 6 summarises the key

findings of this work and provides recommendations for future research.

1.1. References

1. Hall-Stoodley, L., Costerton, J. W. & Stoodley, P. Bacterial biofilms: from the

natural environment to infectious diseases. Nat. Rev. Microbiol. 2, 95–108

(2004).

2. Mattila-Sandholm, T. & Wirtanen, G. Biofilm formation in the industry: A

Chapter 1

5

review. Food Rev. Int. 8, 573–603 (1992).

3. Barnes, R. J. et al. Nitric oxide treatment for the control of reverse osmosis

membrane biofouling. Appl. Environ. Microbiol. 81, 2515–2524 (2015).

4. Coetser, S. E. & Cloete, T. E. Biofouling and biocorrosion in industrial water

systems. Crit. Rev. Microbiol. 31, 213–232 (2005).

5. Balcazar, J. L., Subirats, J. & Borrego, C. M. The role of biofilms as

environmental reservoirs of antibiotic resistance. Front. Microbiol. 6, 1–9 (2015).

6. Baquero, F., Martínez, J.-L. & Cantón, R. Antibiotics and antibiotic resistance in

water environments. Curr. Opin. Biotechnol. 19, 260–265 (2008).

7. Bridier, A., Briandet, R., Thomas, V. & Dubois-Brissonnet, F. Resistance of

Bacterial Biofilms to Disinfectants: a review. Biofouling 27, 1017–1032 (2011).

8. Lee, W. H., Wahman, D. G., Bishop, P. L. & Pressman, J. G. Free chlorine and

monochloramine application to nitrifying biofilm: comparison of biofilm

penetration, activity, and viability. Environ. Sci. Technol. 45, 1412–1419 (2011).

9. Pressman, J. G., Lee, W. H., Bishop, P. L. & Wahman, D. G. Effect of free

ammonia concentration on monochloramine penetration within a nitrifying

biofilm and its effect on activity, viability, and recovery. Water Res. 46, 882–894

(2012).

10. Bal Krishna, K. C., Sathasivan, A. & Chandra Sarker, D. Evidence of soluble

microbial products accelerating chloramine decay in nitrifying bulk water

samples. Water Res. 46, 3977–3988 (2012).

11. Maestre, J. P., Wahman, D. G. & Speitel, G. E. Monochloramine cometabolism

by Nitrosomonas europaea under drinking water conditions. Water Res. 47,

4701–9 (2013).

12. Maestre, J. P., Wahman, D. G. & Speitel, G. E. Monochloramine cometabolism

by mixed-culture nitrifiers under drinking water conditions. Environ. Sci.

Technol. 50, 6240–6248 (2016).

Chapter 1

6

13. Gupta, P., Sarkar, S., Das, B., Bhattacharjee, S. & Tribedi, P. Biofilm,

pathogenesis and prevention—a journey to break the wall: a review. Arch.

Microbiol. 198, 1–15 (2015).

14. Orman, M. A. & Brynildsen, M. P. Persister formation in Escherichia coli can be

inhibited by treatment with nitric oxide. Free Radic. Biol. Med. 93, 145–154

(2016).

15. Arora, D. P., Hossain, S., Xu, Y. & Boon, E. M. Nitric Oxide Regulation of

Bacterial Biofilms. Biochemistry 54, 3717–3728 (2015).

16. Schairer, D. O., Chouake, J. S., Nosanchuk, J. D. & Friedman, A. J. The potential

of nitric oxide releasing therapies as antimicrobial agents. Virulence 3, 271–279

(2012).

17. Yang, Y., Qi, P., Yang, Z. & Huang, N. Nitric oxide based strategies for

applications of biomedical devices. Biosurface and Biotribology 1, 177–201

(2015).

18. Privett, B. J., Broadnax, A. D., Bauman, S. J., Riccio, D. A. & Schoenfisch, M.

H. Examination of bacterial resistance to exogenous nitric oxide. Nitric Oxide 26,

169–173 (2012).

19. Njoroge, J. & Sperandio, V. Jamming bacterial communication: New approaches

for the treatment of infectious diseases. EMBO Mol. Med. 1, 201–210 (2009).

20. Hwang, S. & Meyerhoff, M. E. Polyurethane with tethered copper(II)-cyclen

complex: preparation, characterization and catalytic generation of nitric oxide

from S-nitrosothiols. Biomaterials 29, 2443–2452 (2008).

21. Yang, Y. et al. Development of nitric oxide catalytic coatings by conjugating 3,3-

disulfodipropionic acid and 3,3-diselenodipropionic acid for improving

hemocompatibility. Biointerphases 10, 04A303 (2015).

22. Wu, Y. & Meyerhoff, M. E. Nitric oxide-releasing/generating polymers for the

development of implantable chemical sensors with enhanced biocompatibility.

Talanta 75, 642–650 (2008).

Chapter 1

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23. Barraud, N., Kelso, M. J., Rice, S. A. & Kjelleberg, S. Nitric Oxide : A Key

Mediator of Biofilm Dispersal with Applications in Infectious Diseases. Curr.

Pharm. Des. 21, 31–42 (2015).

24. Oh, B. K. & Meyerhoff, M. E. Catalytic generation of nitric oxide from nitrite at

the interface of polymeric films doped with lipophilic Cu(II)-complex: a potential

route to the preparation of thromboresistant coatings. Biomaterials 25, 283–293

(2004).

8

Chapter 2.

Literature review

2.1. Biofilms: introduction and properties

Bacterial biofilms can be defined as complex microbial communities or

populations that are embedded in a self-produced matrix commonly known as

extracellular polymeric substance (EPS).1,2 In natural environments, around 99% of

bacteria in the world has been estimated to exist in biofilms.3 Bacterial growth in

biofilm form is preferred compared to the free swimming or planktonic growth mode

because of the advantages that biofilm mode of life offers. For instance, biofilm

formation allows the bacterial cells to survive in a hostile environment against a range

of stressors, such as antibiotics, host immune response, and predator.1,4 The close

proximity between cells, either single-species or multi-species microorganism, mostly

resulted in a stable synergistic interaction.5,6 For instance, ammonia oxidising bacteria,

nitrite oxidising bacteria, and heterotroph bacteria are commonly found to coexist in one

community.7 Ammonia oxidising bacteria gain energy from the conversion of ammonia

to nitrite, where the latter is subsequently used by nitrite oxidising bacteria as an energy

source.8,9 Organic matters, which are the by-product of ammonia and nitrite oxidising

bacteria (commonly known as nitrifying bacteria) metabolism, are then utilised by

heterotroph bacteria.7

Chapter 2

9

Biofilm formation is a complex process consisting of several steps (Figure 2.1)

and is affected by different factors, such as the bacteria strains and environmental

parameters.10,11 In general, the process started with diffusion and adsorption of ions and

soluble components such as sugar, proteins, lipids, fatty acids, DNA, and humic acid

onto surfaces creating conditioning film that aids in bacterial attachment.3,12 For

instance, adsorption of divalent cation on anionic substrates will enhance bacteria

attachment by reducing electrostatic repulsion between the surface and bacteria.12 Then,

attachment of planktonic bacteria onto the surface (adhesion) or to each other (cohesion)

would soon follow.11,13,14 The formation of conditioning film and the subsequent

adhesion and attachment of bacteria are regulated by surface factors such as texture,

functionality, charge, hydrophobicity, van der Waals forces, and double layer

interactions.10,12,13 Rougher surface will provide more area for adhesion and are more

difficult to clean, resulting in rapid regrowth of biofilm by multiplication.15 Bacteria

cells, which have a net negative charge, will attach rapidly to the positively charged

surface, while attachment to negatively charged surface is not stable due to the repulsive

forces of the charge.12,16 Bacteria cells prefer to attach to hydrophobic surfaces because

water displacement from the surfaces will promote close contact between cells and the

surfaces.12 However, bacteria attachment to a hydrophilic surface also has been

reported, especially if the surface tension of the bacterial cell wall is higher than the

surface tension of the surrounding liquid.12 Furthermore, it has been shown that initial

adhesion started at locations where the cells are sheltered against shear force, which

facilitates the transition from reversible to irreversible attachment.15

Chapter 2

10

Figure 2.1 Biofilm life cycle.

Cell appendages that help in bacteria motility, such as pili and flagella, also play

an important role in bacterial adhesion to surfaces and formation of biofilm, especially

in Gram-negative species.14 It has been shown that a flagellar mutant of Pseudomonas

aeruginosa is unable to colonise surfaces efficiently, while Escherichia coli bacteria

needed the aid of type I pili and curli fimbriae.11 However, it does not necessarily mean

that bacteria species that do not have cell appendages cannot form a biofilm. Instead,

protein and polysaccharides mediate the attachment of non-motile cells, such as

Staphylococcus epidermidis and Staphylococcus aureus.

When physical appendages of bacteria (such as flagella, fimbriae, and pili) are

able to overcome the repulsive force of the surface, the attachment becomes permanent

or irreversible.3,13 This irreversible attachment is also marked by the secretion of EPS

that aids the adhesion between cells and surface starts.10,12 Moreover, increased

resistance towards antimicrobial agents is observed.13

After the initial lag phase has passed, rapid growth in biofilm population

(exponential phase) begins.3 Biofilm growth could also happen by recruitment of single

cells or cell flocs from the bulk fluid, although the proportion is minor compared to the

replication.11 In the growth step, microbial cells started communicating by producing

Initial

attachment

Irreversible

attachmentGrowth Maturation Biofilm

dispersal

Chapter 2

11

autoinducer signals (commonly known as quorum sensing), such as N-acyl homoserine

lactones (AHL) and autoinducer-1 (AI-1) for gram-negative bacteria, and oligopeptides

for gram-positive bacteria.13,17 When the microbial population reached a certain number,

these autoinducers trigger gene expression that is associated with the adhesive needs of

the biofilm mode of life.3,13 For instance, motility is suppressed and production of

surface appendages is not necessary.3,11 Quorum sensing also modulates other cellular

functions such as pathogenesis, nutrient acquisition, conjugation, motility and secondary

metabolite production.12 At the same time, significant production of EPS is

expected.11,12

Biofilms are mainly comprised of EPS, which can occupy up to 75-90% of the

biofilm total volume, while cells represent only 10-25% of the volume.3,18 EPS can be

found both on the outside and the inside of biofilm,19 and usually are differentiated into

tightly bound, loosely bound, and soluble EPS.14 It is mainly composed of

polysaccharides, protein, extracellular DNA, lipids, and water.14,18,20 The composition of

EPS varies depending on the bacteria species, shear forces, temperature, nutrient

availability, and substrate type.18,19 The presence of EPS in a biofilm is very important

as it holds several functions (Table 2.1).

Chapter 2

12

Table 2.1 EPS functions in biofilms.

Function Relevance for biofilms

Adhesion Aids in planktonic cells colonisation and attachment

Aggregation Bridging between cells, temporary immobilisation of bacterial

populations and development of localised high cell densities

Cohesion Mechanical stability of biofilms (in conjunction with multivalent

cations) and determining biofilm architecture

Water retention Maintaining the highly hydrated environment around the

microorganisms, offer protection against desiccation

Protective barrier Displaying resistance to nonspecific and specific host defences

and tolerance to various antimicrobials agents (oxidising or

charged biocides, antibiotics, metallic cations, ultraviolet

radiation, protozoan grazers)

Sorption (sink) of

organic and

inorganic

compounds

Allows the accumulation of nutrients from the environment and

promotes ion exchange, mineral formation, and toxic metal

accumulation, thus contributing in environmental detoxification.

Storing excess carbon in unbalanced carbon to nitrogen

condition. Allows trapping of biologically active molecules,

such as cell communication signals.

Export of cell

components

Release metabolic products

Enzymatic activity Aids in the digestion of exogenous macromolecules, allows the

degradation of EPS for cells release from biofilm structure

Nutrient source Providing carbon, nitrogen, and phosphorus source

Exchange of genetic

information

Facilitates horizontal gene transfer between biofilm cells

Redox activity Acts as electron donor or acceptor Adapted from Flemming and Wingender,2010.18

As the growth increases, biofilms start to create different structures based on the

growth and environmental condition.1 The most common structure is mushroom shaped

structure known as stromatolites with a diameter between tens to hundreds of

microns.1,3 However, in areas where the water flow is strong, biofilm structure is usually

filamentous, forming a streamer structure.1 Additionally, cells inside a biofilm structure

will undergo physiological differentiation based on different factors, specifically

nutrient and oxygen gradient.21 Studies using oxygen profile measurement showed that

oxygen concentration steadily declines as the electrode travel deeper into the biofilm

structure (Figure 2.2), causing slow cells growth and low or no metabolic activity in the

Chapter 2

13

interior of a biofilm.21 On the other hand, metabolic products are more concentrated

inside the biofilm structure.21

Figure 2.2 Representative images of nutrient and metabolic products distribution inside a

biofilm. (a) Metabolic substrate concentration is higher on the interface compared to the

centre, while (b) metabolic product is more concentrated in the centre of the biofilm. (c) A

metabolic intermediate that is both consumed and produced within the biofilm will have

local maxima inside the biofilm. Adapted from Stewart and Franklin, 2008.21

When a small subpopulation does not have access to both nutrient and oxygen,

the cells become inactive or begin to die.21 The localised cell death is followed by cell

lysis which begins the active dispersal event in a biofilm.20 The initial downregulation

of flagella is halted and motile bacteria can be seen again.11 This new planktonic cells

will then migrate to a new surface and start a new cycle of biofilm formation.20

Biofilm dispersal can also be triggered by various internal and external factors.22

For instance, low nutrient availability will disperse Pseudomonas aeruginosa biofilm,

while high nutrient availability will cause Acinetobacter sp. biofilm to spread out and

disperse.20 Change in iron level has been found to disrupt Pseudomonas aeruginosa

biofilms.23 Other cell signals, including D-amino acids and the unsaturated fatty acids

cis-11-methyl-2-dodecenoic acid and cis-2-decenoic acid, were also found to modulate

biofilm dispersal and formation.20,24,25

Importantly, it was found that alteration in the level of intracellular secondary

messenger molecules known as cyclic di-GMP (c-di-GMP) is the central mechanism

involved in regulating many biofilm behaviours, including biofilm dispersal.26–28 It was

(a) Metabolic substrate (b) Metabolic product (c) Metabolic intermediate

Chapter 2

14

found that at a high level of c-di-GMP, cells mainly exist in biofilm phase, while a low

level of c-di-GMP will promote the switch to planktonic or free-floating phase. The

shift of c-di-GMP level depends on the activity diguanylate cyclases for its synthesis

and phosphodiesterases for its degradation. Several factors are known to alter c-di-GMP

levels, such as nitric oxide, glutamate, and temperature shift.26,29,30

Besides active dispersal events, some passive dispersal events can also occur.

Instead of generating motile planktonic cells (swarming dispersal), the generation of

non-motile cell aggregates that shed from the biofilm structure has been reported.1

These aggregates consist of cells with similar phenotype as biofilm cells, thus cannot be

categorised as planktonic bacteria, and they will move to a new surface following the

fluid flow of the environment.1 However, it was also reported that passive dispersal

could involve the movement of a whole biofilm structure across the surface (surface

dispersal) through shear-mediated transport.1

2.2. Issues with biofilms presence and difficulties in its eradication

Biofilm is ubiquitous in the natural and industrial environment, and it can have

both beneficial and detrimental effects. For examples, biofilms are commonly used in

the field of bioremediation, where they can utilise harmful substances for their

metabolic processes.31 They can also be used to degrade environmentally hazardous

chemicals in soil, remove metals and radionuclides contaminants, treat oil spills and

nitrogen compounds in water, and purify wastewater in bioreactors.3,14 However,

unwanted biofilm presence have caused problems in many areas, such as medical,

transportation, food processing plant, pulp and paper industry, and industrial water

Chapter 2

15

systems.32 For instance, the presence of hospital-related infection, including infection at

the site of implanted medical devices such as artificial prosthetics and catheters, is

linked to the presence of biofilms.3,13 Patients with cystic fibrosis are prone from lungs

infection caused by Pseudomonas aeruginosa biofilm.13 Treatment for biofilm-based

infections can cost more than 1 billion dollars annually.33 Biofilm with a thickness as

small as 25 µm on ship hulls can increase drag by 8%, while biofilm with a roughness

element of 50 µm can increase drag by 22%.31 Biofilm presence in the food industry has

been linked with food poisoning outbreaks,10 and longer sterilisation time requirement.32

In specific, this review will focus on the detrimental effect of biofilms in industrial

water systems.

Biofilms exist in many aspects of the water industry. The presence of biofilm on

membrane filters used in water treatment can clog the pores, causing flux reduction,

transmembrane pressure increase, higher energy consumption, and deterioration in

filtration performance.34 Biofilms cleaning can reduce membrane life significantly, thus

increasing the yearly investment cost.31,34 It was estimated that biofilm problem in

desalination plant worldwide could cost 15 billion US$ per year.31 The formation of

biofilms in a heat exchanger will significantly reduce the heat transfer efficiency, as

convective heat transfer is inhibited and only diffusive heat transfer can occur.35

Moreover, an increase in friction resistance due to biofilm growth will increase energy

consumption, subsequently increasing the cost.35 Biofilm growth on cooling tower

water system may decrease the efficiency to 10% and posed as health risks, as it can

release pathogens into the air.32 Biofilms growth on water-contacting industrial

pipelines can cause corrosion.36 It was estimated that microbial induced corrosion cost

about 16 to 17 billion US$ per year.36 In drinking water industry, biofilms presence can

reduce water quality and act as a reservoir for pathogenic bacteria.32,37 In order to

Chapter 2

16

prevent the occurrence of pathogens and biofilms, disinfectants such as chlorine or

chloramine are commonly added to the water, however this treatment is not necessarily

able to eradicate biofilms.38 Chloramine, which is preferred compared to chlorine due to

its deeper penetration into biofilms, less formation of toxic by-products, and higher

residual throughout distribution systems, is proven to be ineffective towards the

ubiquitous biofilm of nitrifying bacteria, although it was able to penetrate the biofilm

effectively.39,40 Moreover, it was found that nitrifying biofilms and their soluble

microbial products could accelerate the decay of chloramine, releasing chlorine and

ammonia, where the latter is the main nutrient needed for the growth of nitrifying

biofilms, thus promoting the formation of nitrifying biofilm.41–43

Although biofilm problems have been recognised since a long time ago, the

problems persist. It is believed that the increased tolerance or resistance that biofilm has

shown towards antimicrobials, metal cations, and disinfectants is to blame.38,44 Cells in

a biofilm have different traits that make them difficult to eradicate compared to the

planktonic counterpart.14 For instance, 100 times concentration of disinfectants or other

antimicrobials is needed to eradicate biofilm compared to their planktonic

counterpart.45,46 The treatment of cells with metals can kill planktonic bacteria easily,

however, it will trigger protective mechanism for biofilm.44,47

There are several hypotheses that are proposed for biofilm tolerance to biocidal

agents. The main hypothesis is the barrier of the matrix.1 This mechanism is especially

applicable for reactive (such as chlorine), charged (such as metal cations) and large

biocidal agents (some antibiotics).1 EPS matrix, which is mainly negatively charged, is

able to neutralise the charge of several biocides, such as metal cations, aminoglycosides,

and polypeptides, thus render them harmless.18,46 Moreover, the presence of EPS will

dilute biocidal agents to sub-lethal concentration.1 It was reported that chlorine level

Chapter 2

17

inside a biofilm is only 20-25% of the bulk chlorine concentration, with maximum

penetration of 50 to 100 µm.38,39 Moreover, chlorine and chloramine are susceptible to

reaction with organic matter which is present in the EPS, thus decreasing their

disinfection efficiency.38 EPS is also able to neutralise the enzymatic activity of several

enzymes. For instance, β-lactamase activity against Pseudomonas aeruginosa was

neutralised inside the matrix.1 It was also shown that the biofilm matrix could bind

ciproflaxin, thus reducing its concentration.33 The matrix also acts as a barrier for the

toxicity of metals. Metal ions are bound by components of the matrix, such as

extracellular polymers, cell membranes, and cell walls, thus sequestering the metals and

prevent them from interfering in important metabolic processes.44

Subpopulation with different physiological states that consists in a biofilm

structure will eventually contribute to the overall resistance of biofilm. The exposure to

a sub-lethal concentration of biocides can trigger the development of mutagenesis in a

subpopulation of cells, making them able to resist other biocides.38,46 Bacteria cells in a

biofilm also can undergo adaptive physiological changes in response to the toxic

concentration of metal species.44,47 For instance, biofilm treatment with toxic metal

triggers a protective response by upregulating exopolysaccharide production, while the

same treatment can kill planktonic cells easily.47 Some cells are able to up-regulate

genes for efflux pumps, thus cells can get rid of toxic molecules without harming the

cells.38 These factors/genes contributing to resistance can be easily transferred to other

cells due to the high cell density inside biofilm structure.48 Consequently, it was

reported that resistant genes could be detected in an environment where the parent

resistant genes are not available. For instance, vancomycin-resistant genes were

detected in drinking water systems where vancomycin-resistant enterococci are absent.48

Chapter 2

18

These will hence lead to the appearance of more biofilms that are capable of resisting

biocides.

As mentioned before, some cells in a biofilm do not have access to oxygen due

to the concentration gradient inside biofilm structure, leading to the presence of bacteria

with halted or slow metabolism.33,46 Slow growing bacteria have been shown to be more

resistant to biocides than fast growing bacteria.33 It is most likely attributed to the mode

of action of antibiotics that are only effective against bacteria with a certain degree of

activity, due to the antibiotics mechanism that only disrupts microbial processes, such as

cell wall synthesis.1,46 Moreover, some antibiotics are not effective in anaerobic

environment or at acidic pH, due to the accumulation of bacteria metabolic waste.13

Slow growing cells in anoxic environment also have increased tolerance to metal ions,

possibly due to the alteration in metal speciation, decreased metabolic ROS production,

or decrease in metal-catalysed Fenton-type reaction which requires oxygen.44

A special group of bacteria commonly known as persisters are 100 to 10000

times more commonly found inside biofilm than planktonic population.38,44 This

subpopulation of bacteria can easily enter a slow growing or starving state with reduced

metabolism during biocides treatment, rendering them highly tolerant to biocides.46,49

When biocides treatment has passed, persisters can switch back to growing state and

repopulate the biofilm (Figure 2.3),21,49 thus causing biofilm-related problem relapse.49

Chapter 2

19

Figure 2.3 Schematic of biofilm problem relapsed due to persister cells. Adapted from

Lewis, 2007.49

The interaction between bacteria, either the same species or cross-species inside

a biofilm also contributes to biofilm resistance.38,50 For instance, it has been shown that

Pseudomonas aeruginosa cells inside a biofilm can inactivate the antibiotics tobramycin

and gentamicin by secreting a periplasmic cyclic glucan that will complex with the

antibiotics.17 Another mechanism that has been suggested is the chemical interaction

between EPS from each species produce a highly viscous matrix, which will limit the

internal biocides concentration even further.38

Based on the above, it is believed that biofilm resistance to antimicrobials and

disinfectants is multifactorial, i.e. there is no one specific mechanism that solely

Antibiotic/disinfectants

treatment

Treatment stopped

Persister cells remained

Biofilm and planktonic bacteria

Biofilm repopulation

Chapter 2

20

contributes to biofilm resistance (Figure 2.4).38,46 Moreover, it is generally accepted that

the improper use of disinfectants and antibiotics would trigger the formation of bacteria

with increased resistance toward specific biocides, such as Methicillin-Resistant

Staphylococcus aureus (MRSA). The continuing problem caused by biofilm and

difficulty in eradication have driven the need for novel strategies in combating biofilm

that do not depend on the use of conventional disinfectants or antibiotics and most

importantly, do not trigger bacterial resistance.

Figure 2.4 Schematic of multifactorial biofilm resistance mechanisms.

2.3. Current strategies for biofilm prevention and removal

Antibiofilm strategies generally can be divided into three categories: materials

that can prevent biofilm adhesion onto surfaces, materials that can prevent biofilm

growth or induce cells killing, and materials that can disrupt established biofilms.13

-------

++

++

++

++

++

++

Horizontal

gene transfer

Efflux

pump

Neutralisation of

charged biocideBacteria

with

protective

response

Biocide binding by

EPS components

Persister

population

Reduction in

biocide

concentration

Slow growing

population

Anaerobic

environment

: biocide, antibiotics, or metal cations

Chapter 2

21

2.3.1. Adhesion prevention

This antibiofilm strategy is based on the modification of surface properties to

prevent the initial adhesion of bacteria. Surface wettability is the property that is most

commonly altered in designing biofilm-free surface.51 The typical method is by

incorporation of hydrophilic polymer brush such as poly(ethylene glycol) (PEG) and

poly(ethylene oxide) (PEO).12,52,53 Both of these materials could prevent the attachment

of bacteria due to the formation of water layer near the surface that prevents proteins

from attaching.54,55 However, PEG is known to undergo oxidation especially in the

presence of oxygen and transition metal ions, causing the surface to lose their

hydrophilic property.53,56,57 Another polymers that have been used before are

zwitterionic materials such as poly(sulfobetaine) (pSBMA) and poly(carboxybetaine)

(pCBMA), which have similar efficacy towards resisting protein adsorption as PEG.57

These materials have a strong electrostatically induced hydration layer that creates

superhydrophilic surfaces.53,58

Another way to increase hydrophilic property of a surface is to incorporate

inorganic nanoparticles in the surface, and since nanoparticles may also have biocide

properties, their incorporation can lead to an effective antifouling surface. For instance,

the incorporation of silver nanoparticles onto poly(vinylidene fluoride) (PVDF)

membrane improved the surface hydrophilicity. It was proposed that the silver ions

released from the nanoparticles are adsorbed on the nanoparticles in the form of

hydrated silver ions which increase surface hydrophilicity.59 The use of Mg(OH)2

nanoparticles in PVDF membrane increase the number of –OH groups on the surface,

subsequently increase the hydrophilicity.60

Chapter 2

22

The effect of surface functional groups on protein attachment also has been

investigated, and it was concluded that functional groups that can resist protein

adsorption have four similar characteristics: they contain polar functional groups or

hydrophilic groups, contain hydrogen bond acceptor groups, do not contain hydrogen

bond donor groups, and have no net charge/the overall electrical charge is neutral.61,62

Surfaces with water contact angle greater than 150° (known as

superhydrophobic) have been used as an antibiofilm surface. This material is known to

have fouling release property whereby bacteria attachment is not prevented, however,

the bond between bacteria and surface is weakened, therefore the attached cells are

more easily removed by hydrodynamic shear forces.58 Such materials are inspired by

lotus leaf, which has excellent non-wettability. The topography of lotus leaf consists of

few micrometres tall pillars and spaced approximately 20 µm between each other. These

pillars are covered with smaller scale protrusions (approximately 0.2-1 µm) and wax

(Figure 2.5).63 The properties of lotus leaf inspire the fabrication of fouling release

materials, which can be synthesised by combining micro or nanostructure with low

surface energy materials.64,65 Various methods have been used in producing surface

roughness that acts as the surface nanostructure, such as lithography, template-based

techniques, electrospinning, sol-gel synthesis, layer-by-layer deposition, etching,

chemical vapour deposition, electrochemical deposition, electroless galvanic deposition,

and anodic oxidation.65 Fluorine based chemicals are commonly used as the low surface

energy material.66 However, recent research is more focused on synthesising polymer-

nanoparticles composite, due to the toxic nature of fluorine-based chemical.

Nanoparticles are used to provide surface roughness, while polymer acts as low surface

energy material. Several polymers have been used, such as polystyrene (PS), polyvinyl

Chapter 2

23

chloride (PVC), polymethylmethacrylate (PMMA), poly(methylhydrosiloxane) (PMHS)

or polyethylene (PE).66–68

Figure 2.5 Illustration of lotus leaf topography. Adapted from Chen et al., 2012.63

A material that consists of film with lubricating liquid locked by

micro/nanoporous substrate known as SLIPS (Figure 2.6) has been synthesised and

investigated as antibiofilm surface.69–71 It was shown that SLIPS can reduce the

adhesion of Pseudomonas aeruginosa, Staphylococcus aureus, and Escherichia coli 35

times more effectively compared to PEG surface. The antibiofilm property of SLIPS

originates from the very weak adhesion of bacteria to the fluid interface, therefore

adhered bacteria can be easily removed.52,70

Figure 2.6 SLIPS synthesis. Adapted from Wong et al., 2011.69

The synthesis of SLIPS is based on 3 criteria: (1) higher chemical affinity

between lubricating fluid and solid compared to the affinity between ambient fluid and

solid, (2) stable and complete wetting of the solid by the lubricating fluid and (3)

~20 nm

200 nm – 1 µm

water

Functional or

textured solid

Locked lubricating

liquid

Chapter 2

24

lubricating fluid and ambient fluid are immiscible.70 The liquid repellency of SLIPS is

insensitive of texture geometry but highly dependent of the lubricating liquid.69

Other surface properties can also be altered to reduce bacterial adhesion.71 For

example, the topography of poly(dimethylsiloxane) (PDMS) which consists of different

riblets forming diamond-like shape pattern that was inspired by shark skin was shown to

be able to reduce biofilm formation from Staphylococcus aureus compared to the

smooth PDMS (Figure 2.7).72 Even the presence of uniform rectangular, square, or

cylindrical posts on PDMS surfaces has been shown to reduce bacterial attachment,

although the exact mechanism is not understood yet.73 Materials with high surface

energy were found to have higher attachment rates of ammonia oxidising bacteria,

specifically Nitrosomonas europaea and Nitrosospira multiformis.74

Conflicting findings on the effect of material stiffness on bacterial attachment

have been reported. For instance, attachment of Escherichia coli and Staphylococcus

aureus cells were found to correlate positively with increasing hydrogel stiffness.75

However, a negative correlation was found for Escherichia coli RP437 and

Pseudomonas aeruginosa PAO1 attachment on poly(dimethylsiloxane), i.e. increased

stiffness reduced bacterial attachment.76 The exact mechanism through which material

stiffness affects bacterial attachment is yet to be elucidated.16

Chapter 2

25

Figure 2.7 SEM images of Staphylococcus aureus attachment on smooth (left) and shark

skin inspired (known as Sharklet AFTM) PDMS surfaces on day 0 (A and B), day 2 (C and

D), day 7 (E and F), day 14 (G and H) and day 21 (I and J). Taken from Chung et al.,

2007.72

Although adhesion prevention holds the potential to be applied as an antibiofilm

strategy that will not trigger bacteria resistance, inhibition of adhesion can only be

achieved temporarily. Different species of bacteria have evolved complex mechanisms

that allow attachments in a wide range of environmental condition.14 For instance,

bacteria have been shown to be able to alter the surface interaction through the

Chapter 2

26

production of molecules that modify the physicochemical surface properties.5 Once a

pioneer bacterial species is able to overcome the repulsive force of the surface, then a

conditioning layer, which usually consists of proteins and bacteria cells, will be formed.

This conditioning layer will limit the action of the coating due to the accumulation of

cells on the surface, which will attract further bacterial attachment, and thus inactivates

the antibiofilm action of the surface.

2.3.2. Biofilm cells killing and biofilm maturation inhibitor

In this strategy, biofilm formation can be prevented by utilising novel

antimicrobial attached on surfaces that can kill biofilm cells. This material can be

differentiated into two types, surfaces that can kill cells instantly upon contact (most

commonly known as contact killing surfaces) or materials that can leach or release

antimicrobial agent that will kill the surrounding cells.73

One example of “contact-killing surface” uses cationic antimicrobials (such as

quaternary ammonium compounds or alkyl pyridium) as the active biocide, where they

can disrupt cytoplasmic membrane and release cellular content upon contact.77,78

However, the use of quaternary ammonium compounds have been proven to trigger

bacteria resistance,78,79 which is the main problem in utilisation of cell killing in

antibiofilm material. Copper and copper oxide surfaces also possess contact killing trait

and hospital trials that have been conducted showed reduction in surface

contamination.80–82

Modification in surface topography has also been reported to be able to kill

bacteria upon contact. Cicada wings are able to induce cracks in a Pseudomonas

Chapter 2

27

aeruginosa cell that leads to bacterial death solely based on the structure of cicada

wings, composed of nanopillars or nanorods, usually 200 nm tall and spaced 170 nm

apart from centre to centre. Upon contact with Pseudomonas aeruginosa cells, the

nanopillars penetrate and cause cracks on the bacterial cell wall. Eventually, the cells

rupture and sink between the nanopillars (Figure 2.8).83 However, these lysed cells will

aid in the formation of conditioning layers, which, as mentioned before, will aid in the

attachment of other cells and biofilm formation.

Figure 2.8 Cell rupture mechanism by cicada wings. Taken from Ivanova et al., 2012.83

Antibiofilm surfaces that depend on antimicrobial/biocide agent release can be

achieved either physically by soaking or impregnating the carrier material in biocides

(adsorption), or chemically by covalent attachment of the biocides with the surface.84,85

Antimicrobial peptides, which have been shown to exhibit wide antimicrobial action

towards bacteria and fungi and believed not to trigger resistance response, have been

covalently attached to stainless steel or polymer films.84,85 However, it has been

reported that attachment of antimicrobial peptides onto surfaces severely depleted its

a

b

c

d

Chapter 2

28

effectivity, due to the inability of the peptide to interact with cell membrane, and

subsequently, inability to rupture the cell membrane, which is the main mechanism

employed for cell killing by peptides.85

Overall, the long-term benefit of contact killing strategy is tricky as the surface

quickly becomes covered with dead bacteria, which will provide conditioning films for

other bacteria to attach and form biofilms, thus limiting the effect of any active

surface.14,86 For materials that depend on biocide leaching, the limited reservoir of

biocides and difficulty in controlling the leaching kinetic must be considered.

Insufficient biocide release will not be effective in eradicating the biofilms and might

trigger the formation of more resistant biofilms instead. Therefore, novel methods that

can prevent biofilm maturation but not necessarily by cell killing and do not trigger

bacterial resistance are needed.

In recent years, the application of metal-based material or nanoparticles for

antimicrobial application has increased tremendously.53,87 Silver is the most widely

known nanomaterial that exhibit cytotoxicity, however, it has been reported to trigger

bacterial resistance.88 In addition, researchers have focused on the use of metal oxide

nanoparticles because they are cheaper and considered to be non-toxic to human.53,87

Some examples of metal oxide nanoparticles with antibacterial activity are titanium

dioxide (TiO2), silicon dioxide (SiO2), magnesium oxide (MgO), copper oxide (CuO),

and zinc oxide (ZnO).53 Other non-metallic nanoparticles are also known to be

bactericidal, such as carbon nanotubes and graphene.53

Toxicity of metal and metal oxide based surfaces is highly dependent on the

release of the toxic metal ions. For instance, the toxicity of copper and copper oxide

materials depend on the release of toxic Cu2+ or Cu+ ions.81,89 On the contrary,

Chapter 2

29

cytotoxicity of metal and metal oxide nanoparticles is complex and multi-cytotoxic

origins can occur from a single source nanoparticle.87,90 For instance, attachment of

nanoparticles to the bacterial membrane from electrostatic interaction can disrupt

bacterial membrane integrity.90 Bacteria cells are also able to internalise the

nanoparticles, followed by intracellular dissolution of the nanoparticles inside the cells

resulting in further cell damage, known as Trojan horse effect.91 Nanoparticles can

locally change microenvironments near the bacteria, thus producing reactive oxygen

species (ROS) or increasing the solubility of nanoparticles or metal cations.90,92 It has

been reported that copper oxide nanoparticles toxicity against Escherichia coli

originates from complexes leached from the nanoparticles, while the nanoparticles itself

do not play any additional cytotoxic effect to the bacteria.92 However, when zinc oxide

was used, it was found that its toxicity originates not only from the leachate complex

but also the nanoparticles solid itself.91

Figure 2.9 Schematic of antimicrobial mechanisms of metal and metal oxide nanoparticles.

Adapted from Li et al., 200887 and Hajipour et al., 2012.90

ROS

Disruption of

membrane or cell

wall

Generation of reactive

oxygen species

e-

e-

M+ Release of

metal ions

Protein

oxidation

DNA damage

Interruption of

transmembrane

electron transport

Chapter 2

30

It is also known that toxicity of nanoparticles increased with decreasing size of

nanoparticles, as shown for zinc oxide, copper oxide, and silver nanoparticles.92–94 This

is probably due to the fact that smaller sized particles have higher surface charge density

as a result of the increased surface area per unit volume, which therefore imparts more

toxicity from its electrostatic interaction with the negatively charged cell membrane.93

In the case of copper oxide, copper leaching and the subsequent formation of a copper-

peptide complex is responsible for the nanoparticles toxicity, a phenomenon that was

not observed for micrometer-sized copper oxide.92 The interaction between silver

nanoparticles with the bacterial membrane is higher when the nanoparticles size is less

than 5 nm, possibly attributed to easier penetration of the nanoparticle into the cell.94

Morphology of nanoparticles was also found to affect their toxicity. For

instance, octahedral and corner-truncated octahedral cuprous oxide (Figure 2.10a, b and

Table 2.2) is more toxic against Bacillus subtilis compared to Staphylococcus aureus,

Streptococcus faecalis, Pseudomonas aeruginosa, and Enterobacter cloacae.95 On the

contrary, edge-truncated cubic (Figure 2.10j; Table 2.2) showed bacteriostatic effects to

all five bacteria tested. It was hypothesised that the difference in antibacterial activity is

attributed to differences in dominant surface facets ({111} for octahedrons and {100}

for cubes), subsequently exhibiting different adsorption and desorption behaviour

towards the bacteria.95 Similar observation was also reported for silver nanoparticles,

where truncated triangular silver nanoparticles with {111} facet as the dominant facet

have stronger antibacterial effect towards Escherichia coli compared to spherical and

rod-shape silver nanoparticles.96

Chapter 2

31

Figure 2.10 Scanning electron microscopy images of cuprous oxide crystals synthesised at

various condition by glycine-assisted mixed-solvothermal method. Shapes descriptions: (a)

octahedral, (b) corner-truncated octahedral, (c) hexa-cone, (d) hexa-rod, (e) hexa-pod, (f)

poly-cone, (g) polyhedral, (h) polyhedron, (i) cubic structure with truncated edge and/or

concave face-centres, and (j) edge-truncated cubic structure without concave face-centres.

Taken from Pang et al., 2009.95

Table 2.2 Corresponding minimum inhibitory concentrations of different shape cuprous

oxide crystals (refer to Figure 2.10). Taken from Pang et al., 2009.95

Minimum inhibitory concentration (µg/mL)

Shape Bacillus

subtilis

Staphylococcus

aureus

Streptococcus

faecalis

Pseudomonas

aeruginosa

Enterobacter

cloacae

a 12.5 >50 >50 >50 >50

b 6.25 >50 >50 >50 >50

c 6.25 >50 25 >50 25

d 12.5 6.25 25 >50 >50

e 25 >50 >50 >50 >50

f 12.5 >50 >50 >50 >50

g >50 25 >50 25 >50

h 25 25 12.5 >50 >50

i 25 12.5 25 12.5 >50

j 12.5 12.5 12.5 25 6.25

The main drawback in metal and metal oxide based materials is difficulty in

controlling the toxic metal ions release. If the metal ions release is not enough, then it

might trigger the formation of resistant biofilm instead.44 Moreover, metal ions at low

concentrations are needed to support bacterial growth. Metal-based toxicity also relies

on the surrounding condition. For instance, iron coupons exhibit different toxicity in

copper containing Na-HEPES buffer and copper containing Tris-Cl buffer due to

different complexation of copper in the buffer.89 Tris-Cl buffer also showed higher

Chapter 2

32

copper ion release compared to phosphate buffer saline solution.81 It was also reported

that copper oxide nanoparticles might exhibit high toxicity in water that is rich in

organics and low toxicity in freshwater or salty environments due to different copper

leaching behaviour.92

Utilisation of bacteriophage as antibiofilm therapy also have been suggested.97

Bacteriophage are viruses that will inject its genome into host cell’s genome, in this

case, bacteria, causing bacterial cell lysis.98 However, bacteriophages have been shown

to easily diffuse through EPS and produce enzymes that could degrade EPS, such as

depolymerases and dispersin.38,97,99 For instance, phage-induced depolymerases are able

to reduce the formation of Pantoea agglomerans, Serratia marcescens, and

Pseudomonas aeruginosa biofilms.97 Inoculation of ultrafiltration membrane system

with biofilm-forming Pseudomonas aeruginosa, Acinetobacter johnsonii, and Bacillus

subtilis and its corresponding specific phages are able to reduce the number of biofilm

by 40-60% compared the control (bacteria only).100 However, due to the bacteriophage

nature that can only infect a specific type of bacteria, a cocktail of different

bacteriophage is needed to combat multi-species biofilm, thus limiting its

application.97,98 In fact, it was shown that a three-phages cocktail is needed just to

eradicate single species biofilms of Enterobacter cloace.101 Moreover, supplemental

phage addition is sometimes needed due to the regrowth of biofilm after 24 to 48 hours,

as reported before.102 Phage-resistant biofilm was also obtained after the treatment,102

indicating that phage treatment can activate biofilm resistance.

Some acids are recently identified as effective antibiofilm agents. For instance,

acetic acid, either as liquid or dry salts, was found to eradicate Pseudomonas

aeruginosa and Staphylococcus aureus.103,104 The observed antibiofilm activity of acetic

acid is not correlated to pH drop, as a control experiment using hydrochloric acid does

Chapter 2

33

not yield similar effect. Therefore, the inhibition effect observed is caused by the acetic

acid molecules itself.103 Free nitrous acid (FNA), which is the protonated form of nitrite

(HNO2), also has been reported as an antibiofilm agent. It was found to be effective

against many water-related microbial consortiums, such as Nitrosomonas,105

Pseudomonas aeruginosa,106 and Desulfovibrio vulgaris.107 It was also found to be

effective against mixed consortiums of activated sludge,108 sewer biofilms,109 and

reverse osmosis membrane biofilms.110 FNA toxicity mechanism toward bacteria

involves multiple pathways. For instance, treatment of Pseudomonas aeruginosa with

FNA was found to inhibit denitrification activity, which subsequently led to starvation.

At the same time, cell metabolism, protein synthesis, and nucleic acid replication are

halted.111

In recent years, the use of nitric oxide as an antibacterial agent has gathered

attentions. Nitric oxide is known to be toxic against a large number of fungi, bacteria,

viruses, and parasites.112 It is also involved in wound healing regulator and has been

used by eukaryotes in combating bacterial infection.113 Nitric oxide interactions with

reactive oxygen species will produce reactive nitrogen species that can either kill

bacteria (bactericidal) or inhibit bacteria growth (bacteriostatic).112,114,115 Several

mechanisms of nitric oxide antibacterial property have been recognised, such as

interference with DNA replication and cell respiration, inhibition of enzyme function,

and lipid peroxidation.112,115 Although nitric oxide acts by cell killing, few reports have

stated that nitric oxide does not trigger bacteria resistance, which makes nitric oxide

treatment for biofilm control very attractive.113,116 Due to its multiple toxicity

mechanisms, several bacterial mutations have to occur at the same time for the bacteria

to survive,116 therefore, bacterial resistance caused by nitric oxide is difficult to occur.

Chapter 2

34

2.3.3. Biofilm disruption

The final strategy that has been used in eradicating biofilm can be achieved by

disrupting or dispersing (removal) established biofilm. As mentioned before, EPS

consists of different components, such as proteins, polysaccharides, and extracellular

DNA. Therefore, utilisation of enzymes that can degrade specific components of EPS,

such as proteolytic (for protein hydrolysis), polysaccharases (for polysaccharide

hydrolysis), or deoxyribonuclease I (for degradation of extracellular DNA) as

antibiofilm strategy have been reported.98,117,118 The combination of bacteriophages

treatment with EPS degrading enzymes such as dispersin also have been reported.99

However, the use of enzymes as antibiofilm substances are hindered by the limited

enzyme stability and activity in different environmental condition. High temperature

will cause enzymes to denature, while low temperature can decreases enzymatic

activity.98 Enzymes are also sensitive to high salt concentration.98 Change in pH value

also has been reported to alter enzymes efficiency.117 Moreover, free nitrous acid, which

has been mentioned in the previous section, was found to degrade EPS of activated

sludge.108

Another method that has been investigated in biofilm control is to target cellular

communication (quorum sensing), which is a mechanism used by biofilms to coordinate

their communal behaviour, such as biofilm maturation and pathogenesis.13,98,119 Biofilm

maturity can be inhibited by the use of substances that can inhibit quorum sensing,

commonly known as quorum quencher. There are several methods that can be used to

inhibit quorum sensing process, such as reducing the activity of the receptor protein,

inhibition of quorum sensing signal molecule production, degradation of quorum

sensing molecule, and using quorum sensing analogues.119 Quorum sensing analogues

generally have a similar molecular structure with the real quorum sensing species,

Chapter 2

35

therefore it can bind to quorum sensing receptor and inhibit bacterial communication.98

Some of the analogues that have been used as inhibitor are halogenated furanones,

natural furanones and its derivatives, (5-oxo-2,5-dihydrofuran-3-yl)methyl alkanoate, 3-

oxo-C12-(2-aminocyclohexanone), and several polyphenolic compounds produced by

plants, such as vanillin (4-hydroxy-3-methoxybenzaldehyde).98,119 Since furanones are

known to be toxic and carcinogenic for human,120 their application for biofilm control

will be limited.

Energy is required to regulate microbial behaviour, including biofilm formation.

Energy synthesis in cell occurs through the phosphorylation of adenosine diphosphate

(ADP) to adenosine triphosphate (ATP).121 Therefore, substances that can prevent ATP

synthesis, known as chemical uncouplers, has been shown to not only disrupt the

stability of biofilms but can also inhibit biofilm formation and trigger biofilm

detachment.98 Chemical uncouplers are usually weak acids that can dissolve lipid and

carry protons across the cellular membrane. It will enter the cell while carrying

hydrogen ion and deprotonate once inside the cell due to higher pH value. This

deprotonation will deplete the amount of protons available for oxidative

phosphorylation.98 The presence of uncouplers has been shown to reduce the net

synthesis of cellular ATP by 75%, thus reducing the number of extracellular

polysaccharides and protein content in biomass,122 which as mentioned before is the

main protective mechanism employed by biofilm, thus weakening the biofilm structure

and subsequently, the biofilm itself. Chemical uncouplers are also able to cause cell

autolysis by inducing proton gradient loss across the cellular membrane, which

eventually will inhibit microbial attachment or biofilm formation.98 Moreover, the use

of chemical uncouplers can also inhibit biofilm formation through suppression of

quorum sensing substances such as autoinducer-2 (AI-2) and N-acylhomoserine

Chapter 2

36

lactones (AHL).123,124 Some examples of chemical uncouplers are 3,3’,4’,5-

tetrachlorosalicylanilide (TCS), sodium dodecyl sulphate, 2,4-dinitrophenol (DNP), and

dicyclohexylcarbodiimide.98,123,124 However, most of chemical uncouplers are classified

as toxic aromatic compounds,98 thus limiting their application.

As mentioned before (section 2.1), at the end of the life cycle, biofilm would

undergo dispersal to form planktonic bacteria that will able to colonise in a new surface.

Planktonic bacteria are easier to treat compared to biofilm, therefore biofilm removal by

activating dispersal event is an attractive alternative for antibiofilm treatment. Several

substances that have been described in the previous section, such as chemical

uncouplers and bacteriophage, are known to trigger biofilm detachment. Some

substances can cause biofilm dispersal through disruption of intracellular signalling, in

specific through alteration in c-di-GMP level (as described in section 2.1). Because of

their antibiofilm mechanism that exploits cellular signalling pathway, it is believed that

this mode of action will not trigger resistant response.116,125 Such substances are known

to be effective towards a wide spectrum of bacteria and are biodegradable.25 For

example, the fatty acid molecule known as cis-2-decanoic acid was able to induce

dispersal of wide spectrum single species biofilms.24 Some other naturally occurring

substances are also known to be able to induce biofilms dispersal, such as the

supernatant of a marine isolate Bacillus licheniformis that can disperse biofilms of

Micrococcus luteus, Bacillus subtilis, and Escherichia coli.126

Interestingly, low concentration of nitric oxide (below toxic level) was found to

induce biofilm dispersal of various monospecies biofilms, either naturally occurring

biofilms such as Nitrosomonas europaea127 or pathogens such as Pseudomonas

aeruginosa,128 Escherichia coli, and the yeast Candida albicans.129 It was also found to

be effective against multispecies biofilm naturally occurring in water systems34,129 or

Chapter 2

37

wound-relevant pathogenic biofilm.130 Additionally, treatment with nitric oxide was

found to render the biofilm susceptible to antimicrobials.128,131

2.3.4. Nitric oxide as a new antibiofilm agent

Although many strategies have been proposed, it is unlikely that one type of

action could eliminate biofilm successfully. If two different methods are used and each

individual approach is only partially effective, the combination is proposed to be more

efficient.132 For example, the antibiotics aminoglycoside tobramycin and macrolide

clarithromycin have been shown to have moderate activity against biofilms, however,

the combination of the two antibiotics can synergistically eradicate more biofilms.133

The combination of free nitrous acid and hydrogen peroxide can produce the highly

toxic reactive nitrogen intermediate such as peroxynitrite and nitrogen dioxide.134

Consequently, free nitrous acid-hydrogen peroxide combination was found to be highly

effective in the inactivation of anaerobic wastewater biofilms compared to the use of a

single agent. Additionally, it is even more desirable to achieve complete eradication of

biofilm by the combined use of treatments with different modes of action.38 For

example, biofilm treatment with chlorine and bacteriophage is able to reduce biofilm

growth and remove pre-existing biofilm.135 Nitric oxide, which can exert antibiofilm

activity through cell killing (at high concentration, typically mM) or biofilm dispersal

signal (at low concentration, typically nM),114 appears to be advantageous due to its

multi-approach as an antibiofilm agent. As mentioned before, nitric oxide has been

shown to be effective against wide range of bacterial species and it is believed that nitric

oxide will not trigger resistance.116,125 Moreover, control of nitric oxide release is made

easy due to its wide range of active concentration, as it is effective both at high or low

Chapter 2

38

concentration. Biofilm treatment with nitric oxide was also found to reduce the

formation of persisters.136 In fact, nitric oxide is being used by higher level organisms

(including humans) to combat bacterial infections, showing the natural origin of nitric

oxide as antibacterial/antibiofilm agent.137,138 For instance, mice that do not have the

ability to produce nitric oxide endogenously was found to be more susceptible to

microbial infection.139 Therefore, nitric oxide utilisation has emerged as an attractive

strategy for biofilm control.

2.4. Current strategies in nitric oxide utilisation

One of the main challenges in nitric oxide (NO) utilisation as an antibiofilm

agent is in its delivery.113 NO is a free radical with a short half-life and high reactivity,

so its action is only effective as far as 100 µm from its point of origin.112 Therefore,

most research focused on the design and synthesis of materials that can store and release

NO (commonly known as NO-releasing materials), which will be reviewed in the next

section. It should be noted that the majority of literature available on NO-releasing

materials focused on its application in biomedical fields, whereby NO is not only used

as antibiofilm agent but also being used to prevent thrombosis and infection in blood-

contacting biomedical devices.140

2.4.1. Nitric oxide-releasing materials for application in biomedical field

NO-releasing materials generally can be divided into two categories: materials

that are able to adsorb gaseous NO followed by release or materials that release NO via

NO donor pathway. Adsorption of NO gas into delivery vehicles can be achieved by

Chapter 2

39

means of zeolites and metal organic framework. Zeolite-A, which consists of alternating

SiO4 and AlO4 tetrahedra with different metal cations, has been used to chemisorb NO

and was able to release the stored NO gas by exposure to wet gas (humidity).141,142 The

amount of NO that can be stored is dependent on the species and amount of metal

cations that were used, such as cobalt, nickel, copper, manganese, zinc, and

sodium.141,142 Enhancement of NO adsorption can be achieved by utilising metal organic

framework, which has been reported to be able to adsorb 4 times more NO compared to

zeolites.143 This enhancement in NO gas adsorption was correlated to the high density of

coordinatively unsaturated metal cations, such as copper, nickel, cobalt and iron.143

Liposomes, which are vesicles consisting of a liquid core and lipid layers, also

have been used to adsorb gaseous NO.144 NO-loaded liposomes were obtained by

exposing the lipid films, which were synthesised from 1,2-dipalmitoyl-sn-glycerol-3-

ethylphosphocoline, 1-2-dioleoyl-sn-glycero-3-phosphocholine, and cholesterol, to

pressurised NO gas. This process was followed by freezing and thawing to obtain the

final material.144

Nowadays, most NO-releasing materials were formed by exploiting NO donor.

NO donors are molecules that are able to decompose and liberate NO. The most widely

used NO donor for the synthesis of NO-releasing materials is diazeniumdiolates, which

can be synthesised by reacting secondary amines with high pressure of NO gas in the

presence of sodium methoxide or acetonitrile for the deprotonation of amine backbone

and to promote its nucleophilic attack on NO (Scheme 2.1).145,146 Diazeniumdiolates are

able to generate two molecules of NO (thus also known as NONOates) spontaneously in

physiological condition (mostly triggered by pH difference) and the release is not

influenced by biological factors.146,147 As a result, NONOates are the most widely used

NO donor in the synthesis of NO-releasing materials. The NO release profile from

Chapter 2

40

NONOates can be altered by designing structural backbone of the amine-containing

parent molecule (for instance, altering the sizes or substituted cyclic or linear aliphatic

amines) or the hydrogen bonding between NONOates and a primary amine that is

attached to the backbone molecule.140,148 Moreover, NONOates have been modified so

they can liberate NO after contact with a biofilm-related enzyme (β-lactamase) or to

liberate NO and gentamicin (a type of antibiotic) at the same time.149,150 However,

toxicity concerns due to the formation of nitrosamines by-product have been raised.140

Scheme 2.1 NONOates formation and decomposition from secondary amine-bearing

compounds in the presence of sodium methoxide (top) or acetonitrile (bottom). Adapted

from Riccio and Schoenfisch, 2012.146

Another type of NO donor that is widely used is S-nitrosothiols (RSNOs).

Several types of RSNOs are known to be available endogenously in blood and plasma,

such as S-nitrosoglutathione (GSNO), S-nitrosoalbumin (AlbNO), and S-nitrosocysteine

(CysNO).151 Due to the availability of RSNOs in vivo, it is believed that RSNOs are

more biocompatible than NONOates.148,151 A widely used synthetic RSNO known as S-

nitroso-N-acetyl-D-penicillamine (SNAP) is made by the reaction of thiols with

nitrosating agents, such as sodium nitrite.146 In contrary to NONOates, NO release from

RSNOs require triggers, such as thermal or photolysis, metals ions (such as copper or

iron), and reducing agents such as ascorbate or glutathione (Scheme 2.2).146,148

Chapter 2

41

Scheme 2.2 RSNOs formation and decomposition by copper, heat or light, and

glutathione. Adapted from Riccio and Schoenfisch, 2012.146

These different NO sources were then incorporated into different delivery

vehicles to generate NO-releasing materials, either inorganic or organic based. One

example of inorganic NO-releasing nanomaterials is silica-based nanoparticles. Silica

has gathered wide attention due to their ease of preparation, size control, excellent

biocompatibility, and stability.148 NO release can be controlled by modulating the

nanoparticle size, composition, and/or surface hydrophobicity.139 Slomberg et al.

investigated the use of silica nanoparticles with different sizes and aspect ratios but with

a similar amount of overall NO released and reported that particles with decreased size

and increased aspect ratio are more effective against Pseudomonas aeruginosa and

Staphylococcus aureus biofilms due to better NO delivery exhibited by the smaller NO-

releasing particles.152 NO release from sol-gel silica nanoparticles can also be altered by

varying the amine used for NONOates synthesis.153 Amine functionalised silica

nanoparticles which were synthesised from the condensation of tetraethoxysilane

(TEOS) or tetramethoxysilane (TMOS) in the presence of different silane, namely N-(6-

aminohexyl)aminopropyltrimethoxysilane (AHAP3), (aminoethylamino-

Chapter 2

42

methyl)phenethyltrimethoxysilane (AEMP3), or N-(2-aminoethyl)-3-

aminopropyltrimethoxysilane (AEAP3) were found to have different sizes. Conversely,

when the secondary amines were converted to NONOates, the silica nanoparticles

exhibited different NO release amount and half-lives (the time needed to release half of

NO loading).153 A “pre-formation” method, where the formation of NONOates from

aminoalkoxysilanes was done before the formation of NO-releasing silica nanoparticles

also could be employed.154 This method enables silica nanoparticles to exhibit higher

NO payload and NO release (up to 6.3 and 6.7 times greater) compared to the post-

formation method.

Metal nanoparticles have gathered attention as NO-releasing nanocarriers due to

the possibility to trigger the NO release by external stimuli, such as light or magnetic

trigger.146 For example, gold nanoparticles, which exhibited plasmon resonance

property, allow the use of light as the external stimuli.155 Moreover, gold nanoparticles

are inert/biocompatible and can be easily prepared and functionalised, therefore they

have been utilised in various biomedical applications, such as nanocarriers for drug

delivery, as contrast agent, and in nanosensors.148,155 Similar with silica nanoparticles, it

was found that NO release profile from gold nanoparticles is also dependent on the

amine source, where longest half-life was obtained by using ethylene diamine, while

hexadiamine gave the highest NO loading.156 However, it was found that gold

nanoparticles solubility in water is very limited. Thus, functionalisation with tiopronin

was performed, followed by attachment of either diethylenetriamine,

tetraethylenepentamine, or pentaethylenehexamine as the secondary amine.146,156

However, these materials have a very low conversion to NONOates due to the

destabilisation of the tiopronin-containing particles from the base required for

Chapter 2

43

NONOates conversion.156 Platinum-based material that enables the use of light as a

trigger for NO release also has been reported.146

NO-release triggered by magnetic fields could be achieved by employing

superparamagnetic iron oxide nanoparticles (SPIONs).148,156 Contrary to silica and gold

nanoparticles, most reported SPIONs utilises RSNOs as the NO-donor. For instance,

SPIONs which were prepared from the co-precipitation of iron(III) chloride hexahydrate

and iron(II) chloride tetrahydrate with ammonia, were reacted with mercaptosuccinic

acid as thiol source, which was subsequently reacted with sodium nitrite yielding S-

nitrosothiols functionalised SPIONs.156

Although numerous studies have focused on the use of metal-based

nanoparticles, organic-based /polymeric NO-releasing materials make up the majority of

literature. Polymers are preferred due to their encapsulation capability and low

toxicity.147 Incorporation of NO donors onto the polymeric materials can be achieved by

physical process (encapsulation or coating) or chemical process (covalent attachment).

The simplicity of physical process makes it especially attractive for large scale

production. In the physical method, NO donors are non-covalently encapsulated or

embedded within polymeric matrices. For example, PROLI (an example of commercial

NONOates with a half-time of 1.8 s) was incorporated within poly(lactic-co-glycolic

acid) (PLGA) and poly(ethylene oxide-co-lactic acid) (PELA) microparticles.146 RSNOs

such as SNAP also can be encapsulated in PLGA microsphere.157 Biomedical grade

polymers also have been investigated for RSNOs encapsulation, such as silicone

rubber/poly(dimethylsiloxane) (PDMS), Elast-eon E2As (a mixture of PDMS,

poly(hexamethylene oxide) and methylene diphenyl isocyanate), CarboSil

(thermoplastic urethane copolymer, PDMS, and polycarbonate), Tygon (poly(vinyl

Chapter 2

44

chloride) based), and Tecophilic SP-60D-60 (aliphatic hydrophilic polyether-based

polyurethane).158–160 Liposome can be used for encapsulation of Spermine NONOate,

where the release of NO from the lipid vesicle was triggered by temperature and pH

gradient.161

Although physical encapsulation is preferred due to its simplicity, concerns

regarding NO donors premature leaching and toxic nitrosamines release from

NONOates have been raised. Therefore, covalent attachment of NONOates onto

polymeric backbones has been applied to overcome this problem. For examples,

covalent attachment of NONOates to poly(vinyl chloride) (PVC), polyurethane (PU),

silicon rubber, poly(ethylenimine) (PEI), polyester, polymethacrylate, poly(vinyl

alcohol) (PVA) and hydrogels have been reported.140 Polymer nanoparticles, such as

core cross-linked star polymers, have been successfully conjugated with Spermine

NONOates and the subsequent nanoparticle is effective in preventing biofilm

formation.162 Another study synthesised core cross-linked polymer nanoparticles

containing NONOate and gentamicin complex for biofilm eradication. These

nanoparticle complexes were reported to synergistically disperse biofilm and kill

biofilm and planktonic cells at a higher efficiency compared to the combined effect of

gentamicin and NONOates separately.150 Covalent attachment between RSNOs onto

polymer backbones has also been reported.163

Another polymeric vehicle that has been widely employed for NO-releasing

materials is dendrimers. Dendrimers are macromolecules with a spherical shape that

comprises of highly repetitive and branched structure.164 Due to the structure,

dendrimers offer numerous functionalities which, when converted to NONOates or

RSNOs, allow high loading of NO to be stored.165 It was reported that around 2 to 6

µmol NO per mg of dendrimer can be accomplished.140,156 Dual action antibacterial

Chapter 2

45

dendrimer that combines NONOates with quaternary ammonium was reported to be

effective against Pseudomonas aeruginosa and Staphylococcus aureus.166

Although dendrimers enable the synthesis of delivery vehicles with high loading

of NO, its synthesis requires a laborious process. Therefore, micelles, which are core-

shell nanostructures consisting of amphiphilic block copolymers with hydrophilic and

hydrophobic segments, have been proposed as an alternative.148,164 The hydrophobic

inner cores of micelle was proposed to be able to protect NONOates from proton

catalysed NO liberation, thus enhancing its stability.167 GSNO incorporation into the

hydrophobic core was also found to stabilise the NO donor, as the NO release half-time

was extended by 5 times.168 However, as NO donors are mostly hydrophilic, a pre-

treatment step is required in order to load the NO donors into the core.148

Scheme 2.3 NO-releasing materials synthesis strategy in the biomedical field as discussed

in this review.

2.4.2. Application of nitric oxide donors in water industry

Although most research focused on the utilisation and synthesis of NO-releasing

materials for biomedical fields, NO-based treatment does hold the potential to be

applied in the water industry. Barraud et al. reported that the application of 500 nM

NO delivery

Gas

adsorptionNO donors

Zeolites and

metal organic

framework

Liposomes Inorganic Organic

Physical

encapsulation

Covalent

attachmentSilica Metal

Polymeric

matrixLiposomes Nanoparticles Dendrimers Micelles

Chapter 2

46

sodium nitroprusside (SNP; an example of metal based NO donor) to biofilms formed in

recycle and potable water distribution pipelines led to biofilm dispersal by 47% and

60%, respectively.129 Barnes et al. have reported that the use of the NONOates PROLI

at 24 hours intervals to a laboratory scale reverse osmosis membrane system can reduce

the rate of biofouling by 92%.34 The applications of PROLI daily to a membrane

bioreactor with high transmembrane pressure for 37 days (out of 155 days of operation)

are able to reduce the fouling resistance by 56%.169 When PROLI was applied since the

commencement of 85 days operation, a reduction of 32.3% in transmembrane pressure

and delay in biofilm bacterial community development was observed.169 Although

reports on the application of NO and NO-based materials in the water industry are

limited, the existing results suggest a high potential for NO application in water

industry.

2.5. Summary

In this review, biofilm life cycle, which started by attachment of cells onto

surfaces and ended with dispersal event, was presented. Biofilm formations have mostly

negative implications throughout many different fields, including medicine and

industrial water system. Microbial biofilms are ubiquitous in nature and their increased

resistance towards conventional antimicrobial agents and disinfectants drives the need

to develop novel antibiofilm strategy. In general, antibiofilm strategy can be divided

into 3 categories depending on the mode of action, with strategy combining several

modes of action is preferred due to its higher chance of success. NO, which exerts

antibiofilm activity either by cell killing at high concentration or dispersing biofilm at

low concentration, has emerged as an attractive solution. Due to its reactivity, NO

Chapter 2

47

utilisation depends on the synthesis of materials that can store and release NO at the

point of problem. Most NO-releasing materials are focused on the encapsulation or

conjugation of NO donor. The uses of NO donor against biofilms in membrane systems

and water pipelines have shown a high potential of NO to be applied as a new biofilm

eradication method in the water industry. However, daily dosing of NO donors such as

PROLI into industrial water systems is costly and not feasible as it may release NO

prematurely. In addition, most NO-releasing materials that are mentioned in this review

only have limited NO release longevity depending on the amount of NO that can be

adsorbed or loaded during synthesis. Thus, a better alternative would be to develop a

catalytic technology that is able to release NO continuously by converting endogenous

NO source, for instance, nitrite in water. Moreover, this technology will allow the

capability to control the timing and amount of NO released. Therefore, the application

of such materials, commonly known as NO-generating materials, in controlling water-

related biofilms, in specific nitrifying bacteria, would be the focus of this thesis.

2.6. References

1. Hall-Stoodley, L., Costerton, J. W. & Stoodley, P. Bacterial biofilms: from the

natural environment to infectious diseases. Nat. Rev. Microbiol. 2, 95–108

(2004).

2. Yang, L. et al. Combating biofilms. FEMS Immunol. Med. Microbiol. 65, 146–

157 (2012).

3. Garrett, T. R., Bhakoo, M. & Zhang, Z. Bacterial adhesion and biofilms on

surfaces. Prog. Nat. Sci. 18, 1049–1056 (2008).

4. Shrout, J. D., Tolker-Nielsen, T., Givskov, M. & Parsek, M. R. The contribution

of cell-cell signaling and motility to bacterial biofilm formation. MRS Bull. 36,

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48

367–373 (2011).

5. Rendueles, O. & Ghigo, J. M. Multi-species biofilms: How to avoid unfriendly

neighbors. FEMS Microbiol. Rev. 36, 972–989 (2012).

6. Flemming, H.-C. in Marine and Industrial Biofouling 3–12 (2009).

7. Okabe, S., Satoh, H. & Kindaichi, T. in Methods in Enzymology 496, 163–184

(Elsevier Inc., 2011).

8. Rittmann, B. E. & Manem, J. a. Development and experimental evaluation of a

steady-state, multispecies biofilm model. Biotechnol. Bioeng. 39, 914–922

(1992).

9. Daims, H. & Wagner, M. In situ techniques and digital image analysis methods

for quantifying spatial localization patterns of nitrifiers and other

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applications of biomedical devices. Biosurface and Biotribology 1, 177–201

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152. Slomberg, D. L. et al. Role of size and shape on biofilm eradication for nitric

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153. Shin, J. H., Metzger, S. K. & Schoenfisch, M. H. Synthesis of nitric oxide-

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a nitric oxide delivery scaffold. Chem. Mater. 20, 239–249 (2008).

155. Duong, H. T. T. et al. Functional gold nanoparticles for the storage and

controlled release of nitric oxide: applications in biofilm dispersal and

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156. Quinn, J. F., Whittaker, M. R. & Davis, T. P. Delivering nitric oxide with

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co-glycolic acid) microspheres loaded with S-nitroso-N-acetyl-D-penicillamine

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158. Brisbois, E. J., Handa, H., Major, T. C., Bartlett, R. H. & Meyerhoff, M. E.

Long-term nitric oxide release and elevated temperature stability with S-nitroso-

Chapter 2

61

N-acetylpenicillamine (SNAP)-doped Elast-eon E2As polymer. Biomaterials 34,

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159. Wo, Y. et al. Origin of Long-Term Storage Stability and Nitric Oxide Release

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161. Tai, L. A., Wang, Y. C. & Yang, C. S. Heat-activated sustaining nitric oxide

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163. Gierke, G. E., Nielsen, M. & Frost, M. C. S-Nitroso-N-acetyl-D-penicillamine

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Chapter 2

62

polymeric nanoparticle carriers. Chem. Commun. (Camb). 49, 4190–4192 (2013).

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bioreactors. Microb. Biotechnol. 8, 549–560 (2015).

63

Chapter 3.

Nitric oxide generation from nitrite

and ascorbic acid solution in the

presence of copper complex

catalyst

(This chapter is based on the work published in ACS Applied Materials and Interfaces

volume 7, issue 40, page 22148-22156, 2015.)

3.1. Introduction

Materials that are capable of delivering nitric oxide (NO) can be divided into

two types: NO-releasing materials (as reviewed in section 2.4) and NO-generating

materials.1–3 NO-generating materials appear more advantageous than NO-releasing

materials due to their ability to generate NO from endogenous sources, such as S-

nitrosothiols (RSNOs) species which are present in plasma and blood, or from nitrite

ions which are present in water.4,5 Such materials are usually copper or selenium(II)

(organoselenium) based, which can catalyse the decomposition of endogenous NO

donor to form NO.5,6 Other transition metals, such as iron(II), cobalt(II), nickel(II), and

Chapter 3

64

zinc(II), were also found to mediate NO release from S-nitroso-N-acetyl-D-

penicillamine (SNAP; a synthetic RSNOs), although at a significantly lower level

compared to copper.7 Out of all the metals reported, copper is especially attractive due

to its antibacterial activity, thus enhancing its potential as antibiofilm surface.8

The ability of copper to catalyse the reduction reaction of nitrite to NO is known

to occur in bacterial system by nitrite reductase enzyme, in specific blue copper nitrite

reductase.9 This enzyme consists of ligated copper(II) ion centre that can be reduced to

the active copper(I) ion centre, which then provides the electron for nitrite reduction.10,11

Incorporation of redox active copper species could happen by different strategies. For

instance, metallic copper nanoparticles doped inside a polymeric film can undergo slow

corrosion, thus releasing soluble oxides and other copper salts that provide a low level

of active copper(II)/copper(I) ions. These copper ions then interact with endogenous NO

sources in blood to release NO.12–14 Copper nanoparticles incorporated in

collagen/catechol thin films were shown to generate enough NO for inhibiting platelet

activation, thus showing potential application for vascular devices.15 Copper-based

metal organic framework deposited on a flexible polymer also have been used as NO-

generating materials.16 Boes et al. reported copper-containing zeolites that were used to

simultaneously store gaseous nitric oxide and catalytically generate NO from nitrite

(simultaneous NO-storing and generating material).17,18 However, utilisation of zeolites

and metal organic frameworks as NO-releasing/generating materials is not preferred.

This is mainly due to potential toxicity issue of the materials which would be a concern

for the medical application intended for their work.19 The most commonly used strategy

is by employing copper(II) complexes within a polymeric matrix that can be reduced to

copper(I) species in the presence of reducing agents, such as thiols and ascorbate, and

generate NO at the polymeric interface. Oh and Meyerhoff tested the activity of four

Chapter 3

65

different copper complexes in generating NO from sodium nitrite and ascorbate

solution.10 Out of the four complexes tested, Cu-cyclen complex and CuDTTCT

(copper(II)-dibenzo[e,k]-2,3,8,9-tetraphenyl-1,4,7,10-tetraaza-cyclododeca-1,3,7,9-

tetraene) complex exhibited the highest activities, which were found to be comparable

to the catalytic activity displayed by free copper(II) ions. Thus, covalent attachment

between Cu-cyclen within medical grade polyurethane as catheter coatings was

performed.1,20,21 Incorporation of CuDTTCT, which is more preferred over Cu-cyclen

due to its lower solubility in water, into polycarbourethane and poly(vinyl chloride)

(PVC) has been shown to generate NO in the presence of sodium nitrite-ascorbate

solution.10,22 Alternately, cathodic potentials have been used as an alternative means to

reduce copper(II)-tri(2-pyridylmethyl)amine (Cu-TPMA) inside a silicone rubber

catheters to generate NO from a nitrite reservoir.23,24

Herein, the ability of NO-generating material in suppressing biofilm formation

and removing pre-formed biofilm, either on the active material or the nearby surfaces

(the wells) where biofilm was grown, was investigated. CuDTTCT (Scheme 3.1) was

selected as the active catalytic site due to its compatibility with PVC, which is

commonly used as pipeline material for industrial water distribution. Nitrifying bacteria,

an environmentally and industrially relevant chemoautotroph microorganism frequently

found in water distribution systems, are chosen as a model organism.

Chapter 3

66

Scheme 3.1 CuDTTCT structure.

3.2. Experimental methods

3.2.1. Synthesis of copper dibenzo[e,k]-2,3,8,9-tetraphenyl-1,4,7,10-tetraaza-

cyclododeca-1,3,7,9-tetraene complex (CuDTTCT complex) and its

characterisation

Dibenzo[e,k]-2,3,8,9-tetraphenyl-1,4,7,10-tetraaza-cyclododeca-1,3,7,9-tetraene

(DTTCT) was synthesised following the method described by Oh and Meyerhoff.10 In

brief, 0.05 mol of benzil (Aldrich, 98%) and 0.05 mol of o-phenylenediamine (Aldrich,

99.5%) were dissolved in ethanol with a few drops of hydrochloric acid (Ajax

Finechem, 32%). The solution was refluxed at 80 ºC for 6 hours and cooled overnight.

On cooling overnight, light brown crystals formed. The precipitated DTTCT crystals

were filtered and washed with ethanol (resulting in a colour change to white crystalline

product) and dried in a vacuum desiccator. The compound was analysed by 1H-NMR

(Bruker Advance III 300MHz). For NMR analysis, 30 mg of dried DTTCT was

dissolved in 1 mL of deuterated DMSO.

Chapter 3

67

The CuDTTCT complex was synthesised by refluxing 0.01 mol of DTTCT and

0.05 mol of copper acetate monohydrate (Ajax APS) in ethanol at 80 ºC for 2 hours.

After cooling to room temperature overnight, washing with cold ethanol and drying in a

vacuum desiccator, a light blue precipitate was obtained, indicating the incorporation of

copper into the DTTCT structure. For NMR analysis, approximately 10 mg of

CuDTTCT was dissolved in 1 mL of deuterated DMSO.

To synthesise CuDTTCT film, CuDTTCT powder (2 mg) was dissolved in a

pre-made PVC solution in tetrahydrofuran (0.3 mL, 66 mg/mL) and sonicated in an

ultrasonic bath. The resulting solution was cast onto round glass cover slips (18 mm

diameter, ProSciTech). Before use, the glass cover slips were washed with dilute nitric

acid, acetone, and ethanol followed by drying overnight at 110 ºC. The films were dried

at 50 ºC for 12 hours and the resulting film was removed from the glass cover slips. The

amount of copper loading per film was analysed using inductive coupled plasma optical

emission spectrometry (ICP-OES, Perkin Elmer Optima 7300). Before analysis,

CuDTTCT film was weighed and then digested using concentrated nitric acid in a

commercially available microwave digestion bomb. From ICP-OES, the amount of

copper loading per film was found to be 2.88 wt%.

The immobilisation of copper complex in PVC matrix was analysed with a field-

emission scanning electron microscopy (SEM; FEI Nova NanoSEM 230) equipped with

an energy dispersive X-ray detector (EDS; Bruker Silicon Drift Energy Dispersive X-

ray detector). Prior to imaging, the samples were attached to 10 mm metal mounts using

carbon tape and sputter-coated with carbon. Data analysis was performed with Esprit

EDS software.

Chapter 3

68

X-ray photoelectron spectroscopy (XPS; ESCALAB250Xi, Thermo Scientific)

was used to determine the oxidation state of copper. Prior to XPS analysis, the

CuDTTCT films were cleaned by brief immersion in toluene to remove any

contaminants on the surface. Reduction was performed by immersing the pre-cleaned

sample in a 5 mM ascorbic acid (Sigma-Aldrich, ≥99%) solution for 5 minutes. All

samples were dried in a vacuum desiccator for two days before analysis.

3.2.2. NO generation measurement

The amount of NO that can be generated from CuDTTCT via nitrite reduction

was analysed amperometrically using Apollo TBR4100 Free Radical Analyser (World

Precision Instrument) equipped with an ISO-NOP 2 mm probe. The system was

calibrated using S-nitroso-N-acetylpenicillamine (SNAP; Sigma) and copper sulphate

solution according to the manufacturer’s protocol. Measurements were performed in

sterile nitrifying bacteria growth medium (ATCC medium 2265, which consists of three

different stock solutions – stock 1 (final composition in the medium mixture): 25 mM

(NH4)2SO4, 3 mM KH2PO4, 0.7 mM MgSO4, 0.2 mM CaCl2, 0.01 mM FeSO4, 0.02 mM

EDTA, 0.5 µM CuSO4; stock 2: 40 mM KH2PO4, 4 mM NaH2PO4, adjusted to pH 8 by

10 M NaOH; stock 3: 4 mM Na2CO3). CuDTTCT films or bare films (without any

CuDTTCT) as control samples were placed in 20 mL glass vials. Each glass vial was

then filled with 10 mL of media and stirred. After stable baseline was observed, sodium

nitrite (Ajax Finechem) and ascorbic acid were added into the solution to a final

concentration of 5 mM each.

Chapter 3

69

3.2.3. Ascorbic acid oxidation measurement

The change in ascorbic acid concentration over time was investigated via

absorbance measurement using via a colorimetric reagent (1,10-phenanthroline-iron(III)

solution) as described by Besada.25 A 10 mL aliquot of nitrifying bacteria growth

medium was added to a 20 mL glass vial containing CuDTTCT film or bare film.

Sodium nitrite and ascorbic acid at a final concentration of 5 mM were then added into

the vial. A 50 μL aliquot of samples were taken every hour and mixed with 1 mL of

colorimetric reagent and aged for 1 min. The resulting mixture was diluted with 4 mL of

water and the absorbance was recorded using UV-Vis spectrophotometer (Varian Cary

300) at 510 nm.

3.2.4. Biofilm suppression assay

Biofilm assays were performed in sterile 12-well plates (Corning). A mixed

inoculum commercially available nitrifying bacteria for aquarium water purification,

comprising Nitrosospira multiformis, Nitrospira marina and Bacillus sp. (Aquasonic

Bio-Culture), was used as a test strain. The mixed inoculum (1 mL) was added into 100

mL of nitrifying bacteria growth medium (ATCC medium 2265) and incubated for 3

days in the dark (30 ºC, 100 rpm). Three-day-old cultures were inoculated into fresh

medium and 2 mL aliquots (OD = 0.008) were added to each well of sterile 12-well

plates. Sodium nitrite and ascorbic acid in varying concentrations (ranging from 0.1 mM

to 10 mM) were added to each well. For the experiment where CuDTTCT film was

used, the CuDTTCT film was added into each well, followed by the addition of the

sodium nitrite and ascorbic acid solution. The plates were then incubated for 3 days in

the dark (30 ºC, 100 rpm).

Chapter 3

70

The amount of biomass (both planktonic and biofilm cells) was determined

using protein analysis via bicinchoninic acid method (BCA assay; Sigma). To quantify

the growth of planktonic cells, the medium was removed and centrifuged (12,000 rpm,

15 min) to recover the cells. The supernatants were removed and filtered through a 0.2

µm membrane to analyse the extent of copper leaching by ICP-OES. Two washing

stages with phosphate-buffered saline (PBS; Oxoid) were used to remove traces of

ascorbic acid from the planktonic cells. For biofilm measurements, the wells were also

washed twice with PBS to remove traces of ascorbic acid and loosely attached cells.

The BCA working reagent (2 mL) was added into each of the wells that contained

biofilm and to the centrifuge tubes that contained planktonic bacteria. All wells and

tubes were incubated for 30 minutes (37 ºC, 100 rpm) and measurements of the optical

density were performed at 562 nm (Infinite M200 Pro, Tecan). The presence of

CuDTTCT film did not interfere with the assay, as the use of CuDTTCT film in the

absence of bacteria did not result in colour change of the BCA working reagent, even

after the incubation step. Standards solution using Bovine Serum Albumin as per

manufacturer’s instruction was also used for each experiment.

Confocal laser scanning microscopy (CLSM; Olympus FluoViewTM FV1000)

with LIVE/DEAD staining was performed to quantify the bacteria surface coverage and

determine the physiological state (live vs dead) of the adherent bacterial cells. The

culture aliquots (2 mL each) with OD = 0.008 were grown in 35 mm culture dishes with

a coverglass bottom (internal glass diameter 22 mm, ProSciTech) with or without the

presence of bare film, CuDTTCT film, sodium nitrite or ascorbic acid and incubated in

the dark for three days at 30 ºC and 100 rpm. At the end of incubation, the medium was

removed and the dishes were washed twice with PBS. Adhered cells were stained with

400 µL of staining solution containing 3.34 µM of SYTO-9 and 19.97 µM of propidium

Chapter 3

71

iodide in PBS (LIVE/DEAD BacLightTM Bacterial Viability Kits L-7007, Molecular

Probes Inc.) where green stains denote viable bacteria and red or yellow stains denote

non-viable bacteria. The samples were incubated at room temperature for a minimum of

15 minutes. Surface coverage analysis from 12 images across the glass bottom was

performed on live cells (green channel) using image analysis software (Fiji/ImageJ). All

statistical analysis was performed using one-way ANOVA followed by Dunnett post

hoc analysis on Prism (GraphPad).

3.2.5. Biofilm dispersal and metabolic activity assay on pre-formed biofilms

Three-day-old cultures were inoculated into fresh medium and 2 mL aliquots

were added to each well of sterile 12-well plates. A CuDTTCT film was added into each

well and incubated for 3 days in the dark (30 ºC, 100 rpm). One hour before the

incubation period ended, sodium nitrite and ascorbic acid solution were added into each

well, and incubation continued for 1 h. The amount of biomass was then measured using

BCA assay and CLSM analysis as described before.

Adenosine triphosphate (ATP) analysis (Bactiter Glo, Promega) for biomass

metabolic activity measurements was also performed. At the end of 1 h incubation after

the addition of sodium nitrite and ascorbic acid solution, the planktonic bacteria were

collected, centrifuged, washed twice with PBS and resuspended in 100 µL of PBS. For

biofilm analysis, nitrifying biofilm samples were washed twice with PBS and

resuspended in 2 mL of PBS. To detach the cells, the plate was sonicated for 20 minutes

in a sonicating bath. A 100 µL sample of the detached cells was mixed with 100 µL of

the Bactiter Glo working reagent and incubated at room temperature for 5 min. The

luminescence was measured using a microtiter plate luminometer (Wallac Victor2).

Chapter 3

72

3.3. Results and discussion

3.3.1. Characterisation of copper complex (CuDTTCT) powder and films

The obtained spectrum from 1H-NMR spectroscopy analysis of DTTCT ligand

(Figure 3.1) is in accordance with previously reported NMR data.22 After chelation of

the copper(II) ion into the DTTCT complex, the 1H-NMR analysis was performed again

and no structural change in the DTTCT complex framework was observed (result not

shown). SEM and EDS mapping images of CuDTTCT film showed that copper can be

detected uniformly throughout the sample, albeit uneven distribution (Figure 3.2).

However, visual observation of the film does reveal uniform distribution of the copper

complex in the PVC matrix, as the film sample colour change from white (PVC only) to

blue-greenish colour (with the copper complex) without any observable darker coloured

patches. These results subsequently confirmed the presence of copper complex in the

film sample.

Figure 3.1 1H-NMR spectrum of DTTCT in d-DMSO.

8.2 8.0 7.8 7.6 7.4

Chemical shift (ppm)

Chapter 3

73

Figure 3.2 (a) SEM images of CuDTTCT in PVC film and (b) its corresponding elemental

mapping for copper. (c) EDS spectra of CuDTTCT film.

Oxidation state change of copper complex before and after the addition of

reducing agent (ascorbic acid) was analysed by XPS (Figure 3.3). Initially, Cu(II) peak

was detected at 934.8 eV, with two Cu(II) shake up peaks detected at 941.0 and 944.0

eV. The reaction between CuDTTCT with ascorbic acid caused the reduction of

copper(II)-DTTCT to copper(I)-DTTCT, as evidenced by the emergence of Cu(I) peak

at 932.8 eV (Figure 3.3 bottom).

(a) (b)

(c)

Chapter 3

74

Figure 3.3 XPS spectra of CuDTTCT polymer film before (top) and after (bottom)

reduction in 5 mM ascorbic acid solution for 5 min

3.3.2. Nitric oxide generation measurements

In order to validate the role of each chemical species in NO generation from

CuDTTCT-nitrite-ascorbic acid mixture, nitrite (5 mM) was first added into the

CuDTTCT film system, followed by an equimolar amount of ascorbic acid. The

addition of nitrite led to a small increase in NO concentration (Figure 3.4a), while

subsequent addition of ascorbic acid resulted in an abrupt rise in the NO concentration

(up to 80 nM), in good agreement with those obtained by Oh and Meyerhoff.10 It was

found that NO is catalytically formed from nitrite via Cu(II)/Cu(I) redox cycling. In

detail, ascorbic acid reduces Cu(II) to Cu(I), which subsequently reacts with nitrite to

produce NO and, in the process, regenerates Cu(II).10

948 944 940 936 932 928

Co

un

ts (

s-1)

Binding energy (eV)

Cu+

Cu2+

Cu2+

Cu2+

Cu2+

Cu2+

Cu2+

Chapter 3

75

2 Cu2+(DTTCT) + ascorbic acid ⇌ 2 Cu+(DTTCT) + dehydroascorbic acid + 2 H+ (i)

2 Cu+(DTTCT) + 2 NO2– + 2 H2O ⇌ 2 NO + 4 OH– + 2 Cu2+(DTTCT) (ii)

The subsequently formed Cu(II) can be reduced again to Cu(I) following the

proposed Cu(II)/Cu(I) redox cycling mechanism in the presence of excess ascorbic

acid.10,26 Spectrophotometric measurements showed that ascorbic acid can still be

detected in the system even after 7 hours (Figure 3.4b), therefore prolonged generation

of NO was observed.

It has been reported that in acidic conditions, nitrite solutions can form nitrous

acid (HNO2, pKa of 3.2), which can easily decompose to form NO and other nitrogen

oxides.27 As the solution pH remained between 7.3 to 7.6 upon the addition of 5 mM

nitrite-ascorbic acid, it is believed that a major proportion of NO was generated through

the redox cycling pathway, rather than through the acidification of nitrite pathway.27,28

The possible formation of HNO2 in the system which may contribute to the overall NO

release cannot be ruled out, although the system was well-mixed during the addition of

ascorbic acid. A dip in the NO level was also observed in the period between 100 s and

250 s (Figure 3.4a), which can be attributed to the oxidation of the initially generated

NO to nitrite or the oxidation of copper(I) species that lowers the NO signal

momentarily.10 In the absence of CuDTTCT, a maximum of 20 nM NO was generated,

with no prolonged release observed in an equivalent experimental setup. This confirms

the significance of CuDTTCT as an NO generation catalyst.

Chapter 3

76

Figure 3.4 (a) Amperometric measurement of NO generation versus time for control (bare

film) and CuDTTCT film (2.88 wt% of copper) in 10 mL of bacteria medium. The red

arrow indicates the time at which 5 mM of nitrite was added and the blue arrow indicates

the time at which 5 mM ascorbic acid was added. (b) Change in ascorbic acid

concentration in 10 mL of nitrifying bacteria growth medium that contains CuDTTCT

film or bare film (control).

3.3.3. Nitrifying bacteria biofilm suppression by nitrite-ascorbic acid and

CuDTTCT-nitrite-ascorbic acid mixture

The effectiveness of nitrite and ascorbic acid solution on reducing biofilm

formation was studied first in the absence of CuDTTCT. Here, the presence of biomass

was determined by measuring the protein concentration, complemented by confocal

laser scanning microscopy (CLSM) of the LIVE/DEAD-stained biomass. The latter

analysis is to account for the potential detection of biofilm EPS in the protein assay

which would lead to an overestimation of the number of bacteria on the surface.29

In the absence of CuDTTCT, an addition of 5 mM nitrite-ascorbic acid inhibited

the growth of biofilm by 40% and of planktonic biomass by 60% relative to the cultures

that did not receive any treatment (control, Figure 3.5a). Approximately 0.32 mg/L of

leached copper can be detected from this system (Table 3.2). However, it is unlikely

that the 0.32 mg/L of copper leachate detected caused the observed reduction in biofilm

growth, as even 1.5 mg/L of copper ions did not lead to significant biofilm reduction

(a) (b)

0 1 2 3 4 5 6 72

3

4

5

6

Asc. a

cid

co

nce

ntr

atio

n (

mM

)

Time (hours)

control

with CuDTTCT

0 100 200 300 400 500 600 700

-40

-20

0

20

40

60

80

100

120

NO

co

nce

ntr

atio

n (

nM

)

Time (seconds)

control

with CuDTTCT

Chapter 3

77

(Figure 3.7), which will be discussed further later. Therefore, this suggests that the

inhibition effect observed was due to the nitrite-ascorbic acid mixture. Increasing the

amount of nitrite and ascorbic acid to 10 mM led to a severe suppression of the biofilm

formation and only 15% planktonic growth as compared to the untreated controls. The

trend of the biofilm growth suppression was also observed by using LIVE/DEAD

staining in CLSM analysis (Figure 3.5b). The biofilm surface coverage was reduced by

25% after treatment with 1 mM nitrite and ascorbic acid, which is in close agreement

with the ~20% reduction in biofilm biomass measured with the protein assay after the

same treatment. Enhanced reduction in biofilm surface coverage was observed when the

nitrite and ascorbic acid concentration was increased to 5 mM, with only ~30% bacteria

detected on the surface when compared to the control. Furthermore, it was shown that

the biomass remained viable following the nitrite-ascorbic acid treatment in all samples

(Figure 3.5d and e). It appears that nitrite and ascorbic acid in this case inhibit the cell

proliferation of nitrifying bacteria. The suppressed biofilm growth in this study appears

to result from the quick burst of NO generation as observed previously in nitrite-

ascorbic acid system (Figure 3.4).26,30 Such ‘antibiofilm’ activity of the nitrite-ascorbic

acid system is in agreement with the earlier reports, in particular on Gram-negative

pathogens,28,31,32 but no study had been done previously on environmentally-relevant

bacteria, such as nitrifying bacteria.

Chapter 3

78

Figure 3.5 (a) Protein measurements of nitrifying bacteria suppression in nitrite-ascorbic

acid mixture. All values shown are normalised to the protein concentration of control

(inset). Error bars indicate standard error between replicates (n = 3); *p ≤ 0.05 against the

control. (b) Surface coverage analysis of nitrifying biofilms. All values shown are

normalised to the surface coverage of the nitrifying bacteria biofilm in medium only

(control). Error bars indicate standard error between replicates (n = 2); *p ≤ 0.05 against

the control. Confocal laser scanning microscopy images of nitrifying bacteria biofilm

grown (c) in medium only (control) and in the presence of (d) 1 mM and (e) 5 mM of

nitrite-ascorbic acid. Scale bar = 100 µm.

Table 3.1 ICP-OES measurements of copper ions in the bacteria medium after three days

incubation in the presence of bare films

Nitrite and ascorbic acid concentration (mM) Copper ions (mg/L)

0 0.08

1 0.20

5 0.32

(b)

(a) (c)

(d)

(e)

Control

1 mM

5 mM

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

**

*

Rela

tive p

rote

in c

oncentr

ation

Nitrite-ascorbic acid concentration (mM)

Biofilm

Planktonic

Control 1 5 10

*

0

125

250

Pro

tein

co

nce

ntr

atio

n

(µg

/mL

)

Control

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

Rela

tive s

urf

ace c

overa

ge

Nitrite-ascorbic acid concentration (mM)

Control 1 5

*

Chapter 3

79

Recalling the prolonged and 4-fold higher release of nitric oxide (Figure 3.4),

the ‘antibiofilm’ activity of the CuDTTCT-nitrite-ascorbic acid system is investigated

with protein assay-CLSM analyses in the presence of CuDTTCT films. Incorporation of

CuDTTCT into the PVC film alone reduced the planktonic biomass to 32% of the

control culture (Figure 3.6a). This reduction is thought to be the result of copper

toxicity, specifically the leached copper ions (1.5 mg/L, Table 3.2). In order to simulate

the effect of leached copper, copper sulphate at an equivalent concentration was used

(Figure 3.7). In the presence of 1.5 mg/L of copper ions, a 65% decrease of planktonic

bacteria compared to the control was observed with no significant effect towards the

biofilm, which is in accordance with earlier observation in a CuDTTCT-only system.

The toxicity of copper to planktonic bacteria is well documented and may involve

several mechanisms such as oxidative stress induced by Fenton reaction, inactivation of

Fe-S clusters, or lipid peroxidation.33,34

Chapter 3

80

Figure 3.6 (a) Protein measurements of nitrifying bacteria suppression in CuDTTCT,

nitrite and ascorbic acid mixture. All values shown are normalised to the protein

concentration of control (inset). Error bars indicate standard error between replicates (n =

4); *p ≤ 0.05 against the control. (b) Surface coverage analysis of nitrifying biofilms. All

values shown are normalised to the surface coverage of the nitrifying biofilm grown in the

presence of bare film (control). Error bars indicate standard error between replicates (n =

2); *p ≤ 0.05 against the control. Confocal laser scanning microscopy images of nitrifying

biofilm grown in the presence of (c) bare film (control), (d) CuDTTCT film, (e) CuDTTCT

film and 1 mM of nitrite-ascorbic acid, and (f) CuDTTCT film and 5 mM of nitrite-

ascorbic acid. Scale bar = 100 µm.

Table 3.2 ICP-OES measurements of copper ions in the bacteria medium after three days

incubation in the presence of CuDTTCT films.

Nitrite and ascorbic acid

concentration (mM)

Copper ions (mg/L) %mass of copper leached out

of the CuDTTCT films

0 1.48 0.44

1 2.61 0.77

5 3.29 0.94

(a)

(b)

(c)

(d)

(e)

(f)

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

*

*

**

**

Rela

tive p

rote

in c

oncentr

ation

Nitrite-ascorbic acid concentration (mM)

Biofilm

Planktonic

Control 0 0.1 0.5 1 5

*

0

200

400

Pro

tein

concentr

ation

(µg/m

L)

Control

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

Rela

tive s

urf

ace c

overa

ge

Nitrite-ascorbic acid concentration (mM)

Control 0 1 5

*

Control

0 mM

1 mM

5 mM

Chapter 3

81

Figure 3.7 Protein measurements of the nitrifying bacteria after the addition of free

copper ion (in the form of copper sulphate). All values shown are normalised to the

protein concentration of control (inset). Error bars indicate standard error between

replicates (n = 3); *p ≤ 0.05 against the control.

Addition of nitrite-ascorbic acid of up to 1 mM in the presence of CuDTTCT

further reduced the planktonic and biofilm biomass to ~20% and ~43% of the control

value, respectively. Such suppression of the biofilm formation was not observed at the

same nitrite-ascorbic acid concentration in the absence of CuDTTCT. Further increase

of nitrite-ascorbic acid concentration to 5 mM in a CuDTTCT system resulted in more

than 95% reduction of biofilm biomass compared to the control and complete inhibition

of planktonic growth. The addition of 5 mM nitrite-ascorbic acid into the CuDTTCT

system was associated with detection of 3.3 mg/L of leached copper ions (Table 3.2),

which account for less than 1% of copper from CuDTTCT, suggesting the excellent

stability of CuDTTCT inside the PVC matrix. However, this observed increase in

copper leachate could alter water quality and affect the environment. Future research

directed at ligand design that can increase the stability of copper centre and prevent

leaching would be of interest.

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

*

***

Rela

tive p

rote

in c

oncentr

ation

Copper concentration (mg/L)

Biofilm

Planktonic

Control 1.5 2.6 3.3 10

*

0

100

200

300

Pro

tein

co

nce

ntr

atio

n

(µg

/mL

)

Control

Chapter 3

82

The detected 3.3 mg/L of copper ions from the system did not lead to significant

reduction in nitrifying biofilm biomass, although 77% reduction in planktonic biomass

was observed (Figure 3.7). In fact, the presence of 10 mg/L of copper ions was not able

to fully eradicate the biofilm (only 50% reduction compared to control) as opposed to

drastic biofilm reduction observed in the 5 mM CuDTTCT-nitrite-ascorbic acid system.

Therefore, the system’s high inhibition of biofilm formation cannot be solely attributed

to the leached copper ions. Taken together, it can be concluded that biofilm suppression

is predominantly caused by the CuDTTCT-nitrite-ascorbic acid mixture, most likely due

to the activity of catalytically generated NO as a cell proliferation inhibitor. In

agreement with the biofilm inhibition trend that was determined by the protein assay,

CLSM analysis revealed 25% biofilm surface coverage with the CuDTTCT system in

the presence of 1 mM nitrite-ascorbic acid relative to the control (p = 0.058, Figure

3.6b). Further increase of nitrite-ascorbic acid concentration to 5 mM significantly

inhibited biofilm formation (surface coverage reduced to 2%).

Comparing the two different systems that have been investigated, a higher

amount of nitrite and ascorbic acid was needed to achieve comparable biomass

suppression in the absence of CuDTTCT. For instance, 5 mM nitrite and ascorbic acid

in the absence of CuDTTCT were required to reduce the biofilm surface coverage by

~70% relative to the control (determined by CLSM, Figure 3.5b), while lower

concentration of nitrite and ascorbic acid (1 mM) was sufficient to acquire a comparable

biomass reduction in the presence of CuDTTCT (Figure 3.6b). Similar to the nitrite-

ascorbic acid system, the biomass remained viable following the CuDTTCT-nitrite-

ascorbic acid treatment (Figure 3.6e and f), even at the highest concentration tested (5

mM). This implies that NO generated in the CuDTTCT-nitrite-ascorbic acid system acts

as a cell proliferation inhibitor to the nitrifying bacteria. NO can acts as an antimicrobial

Chapter 3

83

agent by several mechanisms, in particular, its reactivity towards transition metals. The

NO reactivity toward iron ions has been known to inhibit bacterial respiration,35 while

the inactivation of zinc metalloproteins by NO inhibits DNA replication.36 The synthesis

and repair of DNA are also inhibited by reactions between NO and tyrosyl radicals

which limits the concentration of the DNA precursor.36

In comparison to the nitrite-ascorbic acid system, it would be reasonable to

deduce that the enhanced bacteriostatic activity of the CuDTTCT-nitrite-ascorbic acid

system is at least in part due to the 4-fold higher nitric oxide release in the quick burst

(first 200 s) along with the subsequent prolonged release of NO. It was shown in earlier

studies that higher amount of NO that was released over short durations is more

damaging to Gram-negative bacteria than a prolonged release of lower amounts of

NO.37 Similar findings were also reported on human fibroblasts, where the reduction in

fibroblast numbers exhibited by the copper-nitrite-ascorbic acid system has higher

degree of correlation to the amount of nitric oxide generated in the quick burst (first 200

s) rather than the total amount of nitric oxide over 600 s.26

3.3.4. Nitrifying bacteria biofilm dispersal upon the addition of nitrite-ascorbic acid

in the presence of CuDTTCT

The effect of NO on a pre-formed biofilm was also investigated by adding nitrite

and ascorbic acid to a CuDTTCT system following 3 days of biofilm growth. The term

“pre-formed biofilm” is used to refer to biofilms that cannot be removed through simple

washing steps alone, although biofilm maturity may not have been reached yet. The

system was found to be effective at dispersing the biofilm even with as little as 0.1 mM

nitrite-ascorbic acid, which resulted in ~40% less biofilm compared to the CuDTTCT-

Chapter 3

84

only system (Figure 3.8a). The addition of nitrite-ascorbic acid of up to 1 mM was

effective at dispersing the biofilm by 40%; however, when the concentration was

increased to 5 mM, the dispersal effect was reduced, with only 22% of biofilm dispersal

detected (p-value = 0.089 against the CuDTTCT film with no nitrite and ascorbic acid

added). Confirming the trend of biofilm dispersal determined by the protein assay,

surface coverage analysis revealed that 1 mM nitrite-ascorbic acid addition can reduce

the biofilm surface coverage by ~50% as compared to the CuDTTCT-only system with

no viability loss observed (Figure 3.8b and e). Although an increase in leached copper

concentration was observed upon the addition of nitrite-ascorbic acid into the system

(Table 3.3), the observed dispersal effect was more attributed to NO generated by the

combination of CuDTTCT-nitrite and ascorbic acid system. The ability of NO (by

addition of a NO donor or NO gas) to cause biofilm dispersal has been reported on a

wide spectrum of microorganisms, including the Gram-positive Staphylococcus

epidermidis,38 Bacillus licheniformis,38 the Gram-negative Pseudomonas aeruginosa,39

Escherichia coli,38 Serratia marcescens,38 Vibrio cholerae,38 Nitrosomonas europaea,40

as well as the Candida albicans yeast.38 In the case of Nitrosomonas europaea, which is

one of the major members of nitrifying microorganisms in aquatic environments, the

switch between biofilm and planktonic states is induced by the regulation of the motility

expression and/or chemotaxis responses by NO, which is potentially the mechanism

adopted by the test organism in this study.40

Chapter 3

85

Figure 3.8 (a) Protein measurements of the nitrifying bacteria upon addition of nitrite and

ascorbic acid on pre-formed biofilm grown in the presence of CuDTTCT. All values

shown are normalised to the protein concentration of the control (inset). Error bars

indicate standard error between replicates (n = 4); *p ≤ 0.05 against the control (bare

film); #p ≤ 0.05 against the CuDTTCT film (0 mM). (b) Surface coverage analysis of

nitrifying biofilms. All values shown are normalised to the surface coverage of nitrifying

biofilm grown in the presence of bare film (control). Error bars indicate standard error

between replicates (n = 3); * p ≤ 0.05 against the control and CuDTTCT film (0 mM).

Confocal laser scanning microscopy images of nitrifying bacteria biofilm grown in the

presence of (c) bare film (control), (d) CuDTTCT film, (e) CuDTTCT film and 1 mM of

nitrite-ascorbic acid, and (f) CuDTTCT film and 5 mM of nitrite-ascorbic acid. Nitrite and

ascorbic acid were added on pre-formed biofilm grown in the presence of CuDTTCT 1

hour before incubation ended. Scale bar = 100 µm.

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

*

**

*

*

*

Re

lative

pro

tein

co

nce

ntr

atio

n

Nitrite-ascorbic acid concentration (mM)

Biofilm

Planktonic

Control 0 1 50.1 0.5

*#*#

*#

Control0

150

300

Pro

tein

co

nce

ntr

ation

(µg

/mL

)

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

Rela

tive s

urf

ace c

overa

ge

Nitrite-ascorbic acid concentration (mM)Control 0 1 5

**

(a)

(b)

(c)

(d)

(e)

(f)

Control

0 mM

1 mM

5 mM

Chapter 3

86

Table 3.3 ICP-OES measurements of copper ions in the bacteria medium after the end of

second incubation step in the presence CuDTTCT films.

Nitrite and ascorbic acid concentration (mM) Copper ions (mg/L)

0 1.90

1 3.34

5 4.54

From the surface coverage analysis, the addition of 5 mM nitrite and ascorbic

acid was also found to be not as effective as for the 1 mM concentration in reducing the

biofilm biomass (Figure 3.8b,e,f). The analysis, however, revealed the presence of dead

(red) cells after addition of 5 mM nitrite-ascorbic acid. In this case, it is likely that the

cell death occurred at a faster rate relative to the biomass dispersal, thereby reducing the

efficacy of biofilm dispersal. The observed toxicity of either the increase in copper

leachate concentration or the NO generated at 5 mM nitrite-ascorbic acid is consistent

with the ATP analysis, as described below.

The level of ATP detected in biofilms grown in the presence of CuDTTCT was

60% lower than the level of ATP detected in control biofilms (Figure 3.9). Similarly,

planktonic bacteria also had 60% less ATP after exposure to CuDTTCT. Interestingly,

although protein and ATP measurements on planktonic bacteria revealed similar trends

for control (bare film) and CuDTTCT film only, a different trend was observed for the

biofilm. While no significant reduction was observed for the biofilm biomass by protein

and surface coverage analysis between the control and CuDTTCT film, 60% lower ATP

was detected in biofilms grown in the presence of CuDTTCT film compared to control

biofilm. This indicates that the presence of CuDTTCT or/and the soluble copper ion

could induce stress in the biofilm bacteria which resulted in a lower amount of ATP

being produced rather than removal of the biofilm biomass. A small and insignificant

reduction in ATP was observed after the addition of 1 mM nitrite and ascorbic acid (p-

value = 0.062 for the biofilm and p-value = 0.074 for the planktonic against the

Chapter 3

87

CuDTTCT film). However, after the addition of 5 mM nitrite-ascorbic acid, ATP levels

were significantly lowered in both biofilm and planktonic bacteria. The decrease in ATP

values in this instance could be attributed to viability loss due to cellular damage from

NO exposure as well as the increase in leached copper concentration that is triggered by

the addition of nitrite and ascorbic acid. In fact, samples containing CuDTTCT and

treated with 5 mM nitrite and ascorbic acid exhibited dead bacteria that were stained

with propidium iodide, which is an indicator of cell membrane disruption. NO is

reported to cause membrane disruption via radical lipid peroxidation.41 The

deterioration of lipid bilayer of the cell membrane could cause the increase in cell

surface roughness and membrane degradation.37 Interestingly, cell death by membrane

disruption was not observed when CuDTTCT and 5 mM of nitrite and ascorbic acid

were added before biofilm was formed (Figure 3.6f). It appears that the mode of action

of this catalytic system may be dependent on the growth stages of the bacteria. When

bacteria are exposed to nitrite, ascorbic acid and copper complex from the beginning of

incubation, i.e. during the lag phase, bacterial cells may have access to energy resources

allowing them to resist potential membrane damage and cell death induced by NO,

while being unable to trigger cell division and proliferation mechanisms. However, in

pre-formed biofilm system, bacterial cells are in a stationary phase without access to

fresh nutrients and have low metabolic activity, thus the addition of nitrite and ascorbic

acid in the presence of copper complex led to cell death by membrane disruption.

Antimicrobial action dependent on the bacterial growth phase has been widely reported

before, including for antibiotics.34,42 For instance, the polypeptide colistin, which can

solubilise the cytoplasmic membrane, was found to be more effective against stationary-

phase, dormant bacteria with low metabolic activity as compared to cells with high

metabolic activity.43 However, it is also possible that the initial nitric oxide burst

Chapter 3

88

generated before the formation of biofilm would induce membrane disruption or

reduction of metabolic activity, but the three days incubation period allowed some

bacterial cells to regain their viability.

Figure 3.9 Luminescence measurements of the nitrifying bacteria upon the addition of

nitrite and ascorbic acid on pre-formed biofilm. All values shown are normalised to the

luminescence of the control. Error bars indicate standard error between replicates (n = 2);

*p ≤ 0.05 against the control (bare film); #p ≤ 0.05 against the CuDTTCT film.

3.4. Summary

A lipophilic copper(II) complex (CuDTTCT) as an NO generating catalyst has

been successfully incorporated into PVC films and tested for the control of nitrifying

bacteria biofilm formation in the presence of nitrite and ascorbic acid. Copper(II) in the

complex was reduced to copper(I) by ascorbic acid, which subsequently reduced nitrite

to NO while being oxidized back to copper(II). Amperometric measurements revealed

an initial surge of NO generated by the copper complex system upon the addition of

ascorbic acid followed by a continuous production of NO. The potential of catalytically

generated NO to control the formation of and disperse a nitrifying bacteria biofilm was

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

*

*

Re

lative

lu

min

esce

nce

Nitrite-ascorbic acid concentration (mM)

Biofilm

Planktonic

Control 0 1 5

*

*#*#

Chapter 3

89

demonstrated. Nitrifying bacteria biofilm biomass was suppressed in the presence of

CuDTTCT-nitrite-ascorbic acid which is predominantly caused by NO. Moreover, the

system is also effective at dispersing established biofilms. No significant dead cells

were observed in the biofilm suppression studies, which indicate that prior to biofilm

formation the NO generated by copper complex-nitrite-ascorbic acid mainly inhibits cell

proliferation. Biofilm dispersal studies showed that the copper complex system with 1

mM of nitrite and ascorbic acid produced sufficient NO to effectively disperse the

accumulated biofilm. At a higher concentration of nitrite and ascorbic acid (5 mM),

biofilm dispersal effect was reduced and significant dead cells were observed. In

summary, this study highlighted the use of copper complex immobilised in PVC and

nitrite-ascorbic acid to minimise formation and disperse nitrifying bacteria biofilms

through sustained generation of NO.

3.5. References

1. Hwang, S. & Meyerhoff, M. E. Polyurethane with tethered copper(II)-cyclen

complex: preparation, characterization and catalytic generation of nitric oxide

from S-nitrosothiols. Biomaterials 29, 2443–2452 (2008).

2. Yang, Y. et al. Development of nitric oxide catalytic coatings by conjugating 3,3-

disulfodipropionic acid and 3,3-diselenodipropionic acid for improving

hemocompatibility. Biointerphases 10, 04A303 (2015).

3. Wu, Y. & Meyerhoff, M. E. Nitric oxide-releasing/generating polymers for the

development of implantable chemical sensors with enhanced biocompatibility.

Talanta 75, 642–650 (2008).

4. Barraud, N., Kelso, M. J., Rice, S. A. & Kjelleberg, S. Nitric Oxide : A Key

Mediator of Biofilm Dispersal with Applications in Infectious Diseases. Curr.

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Pharm. Des. 21, 31–42 (2015).

5. Yang, Y., Qi, P., Yang, Z. & Huang, N. Nitric oxide based strategies for

applications of biomedical devices. Biosurface and Biotribology 1, 177–201

(2015).

6. Wo, Y., Brisbois, E. J., Bartlett, R. H. & Meyerhoff, M. E. Recent advances in

thromboresistant and antimicrobial polymers for biomedical applications: just say

yes to nitric oxide (NO). Biomater. Sci. 4, 1161–1183 (2016).

7. McCarthy, C. W., Guillory II, R. J., Goldman, J. & Frost, M. C. Transition Metal

Mediated Release of Nitric Oxide (NO) from S-Nitroso-N-acetylpenicillamine

(SNAP): Potential Applications for Endogenous Release of NO on the Surface of

Stents via Corrosion Products. ACS Appl. Mater. Interfaces 8, 10128–10135

(2016).

8. Gunawan, C., Teoh, W., Marquis, C. & Amal, R. Cytotoxic origin of copper (II)

oxide nanoparticles: comparative studies with micron-sized particles, leachate,

and metal salts. ACS Nano 5, 7214–7225 (2011).

9. Cutruzzolà, F. Bacterial nitric oxide synthesis. Biochim. Biophys. Acta -

Bioenerg. 1411, 231–249 (1999).

10. Oh, B. K. & Meyerhoff, M. E. Catalytic generation of nitric oxide from nitrite at

the interface of polymeric films doped with lipophilic Cu(II)-complex: a potential

route to the preparation of thromboresistant coatings. Biomaterials 25, 283–293

(2004).

11. Opländer, C. et al. Redox-mediated mechanisms and biological responses of

copper-catalyzed reduction of the nitrite ion in vitro. Nitric Oxide 35, 152–164

(2013).

12. Major, T. C. et al. The hemocompatibility of a nitric oxide generating polymer

that catalyzes S-nitrosothiol decomposition in an extracorporeal circulation

model. Biomaterials 32, 5957–5969 (2011).

13. Wu, Y. et al. Improving blood compatibility of intravascular oxygen sensors via

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catalytic decomposition of S-nitrosothiols to generate nitric oxide in situ. Sensors

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14. Amoako, K. A. & Cook, K. E. Nitric oxide-generating silicone as a blood-

contacting biomaterial. ASAIO J. 57, 539–544 (2011).

15. Luo, R. et al. Copper-Incorporated Collagen/Catechol Film for in Situ Generation

of Nitric Oxide. ACS Biomater. Sci. Eng. 1, 771–779 (2015).

16. Neufeld, M. J., Harding, J. L. & Reynolds, M. M. Immobilization of Metal-

Organic Framework Copper(II) Benzene-1,3,5-tricarboxylate (CuBTC) onto

Cotton Fabric as a Nitric Oxide Release Catalyst. ACS Appl. Mater. Interfaces 7,

26742–26750 (2015).

17. Boes, A.-K., Wheatley, P. S., Xiao, B., Megson, I. L. & Morris, R. E.

Simultaneous and cooperative gas storage and gas production using bifunctional

zeolites. Chem. Commun. (Camb). 5, 6146–6148 (2008).

18. Boës, A.-K., Xiao, B., Megson, I. L. & Morris, R. E. Simultaneous Gas Storage

and Catalytic Gas Production Using Zeolites—A New Concept for Extending

Lifetime Gas Delivery. Top. Catal. 52, 35–41 (2009).

19. Seabra, A. B. & Durán, N. Nitric oxide-releasing vehicles for biomedical

applications. J. Mater. Chem. 20, 1624–1637 (2010).

20. Liu, K. & Meyerhoff, M. E. Preparation and characterization of an improved

Cu(2+)-cyclen polyurethane material that catalyzes generation of nitric oxide

from S-nitrosothiols. J. Mater. Chem. 22, 18784–18787 (2012).

21. Puiu, S. C. et al. Metal ion-mediated nitric oxide generation from polyurethanes

via covalently linked copper(II)-cyclen moieties. J. Biomed. Mater. Res. - Part B

Appl. Biomater. 91, 203–212 (2009).

22. Zhao, H., Feng, Y. & Guo, J. Polycarbonateurethane films containing complex of

copper (II) catalyzed generation of nitric oxide. J. Appl. Polym. Sci. 122, 1712–

1721 (2011).

23. Ren, H. et al. Electrochemically modulated nitric oxide (NO) releasing

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biomedical devices via copper(II)-Tri(2-pyridylmethyl)amine mediated reduction

of nitrite. ACS Appl. Mater. Interfaces 6, 3779–3783 (2014).

24. Ren, H. et al. Thromboresistant/anti-biofilm catheters via electrochemically

modulated nitric oxide release. Bioelectrochemistry 104, 10–16 (2015).

25. Besada, A. A facile and sensitive spectrophotometric determination of ascorbic

acid. Talanta 34, 731–732 (1987).

26. Opländer, C. et al. Characterization of novel nitrite-based nitric oxide generating

delivery systems for topical dermal application. Nitric Oxide 28, 24–32 (2013).

27. Lundberg, J. O., Weitzberg, E. & Gladwin, M. T. The nitrate-nitrite-nitric oxide

pathway in physiology and therapeutics. Nat. Rev. Drug Discov. 7, 156–167

(2008).

28. Carlsson, S., Wiklund, N. P., Engstrand, L., Weitzberg, E. & Lundberg, J. O.

Effects of pH, nitrite, and ascorbic acid on nonenzymatic nitric oxide generation

and bacterial growth in urine. Nitric oxide Biol. Chem. 5, 580–586 (2001).

29. Douterelo, I. et al. Methodological approaches for studying the microbial ecology

of drinking water distribution systems. Water Res. 65, 134–156 (2014).

30. Schairer, D. O., Chouake, J. S., Nosanchuk, J. D. & Friedman, A. J. The potential

of nitric oxide releasing therapies as antimicrobial agents. Virulence 3, 271–279

(2012).

31. Kishikawa, H. et al. Control of pathogen growth and biofilm formation using a

urinary catheter that releases antimicrobial nitrogen oxides. Free Radic. Biol.

Med. 65, 1257–1264 (2013).

32. Firmani, M. & Riley, L. Reactive nitrogen intermediates have a bacteriostatic

effect on Mycobacterium tuberculosis in vitro. J. Clin. Microbiol. 40, 1–6 (2002).

33. Booth, S. C. et al. Differences in metabolism between the biofilm and planktonic

response to metal stress. J. Proteome Res. 10, 3190–3199 (2011).

34. Lemire, J. A., Harrison, J. J. & Turner, R. J. Antimicrobial activity of metals:

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mechanisms, molecular targets and applications. Nat. Rev. Microbiol. 11, 371–

384 (2013).

35. Stern, A. M. & Zhu, J. An introduction to nitric oxide sensing and response in

bacteria. Adv. Appl. Microbiol. 87, 187–220 (2014).

36. Fang, F. C. Antimicrobial reactive oxygen and nitrogen species: concepts and

controversies. Nat. Rev. Microbiol. 2, 820–832 (2004).

37. Deupree, S. M. & Schoenfisch, M. H. Morphological analysis of the

antimicrobial action of nitric oxide on gram-negative pathogens using atomic

force microscopy. Acta Biomater. 5, 1405–1415 (2009).

38. Barraud, N. et al. Nitric oxide-mediated dispersal in single- and multi-species

biofilms of clinically and industrially relevant microorganisms. Microb.

Biotechnol. 2, 370–378 (2009).

39. Barraud, N. et al. Involvement of nitric oxide in biofilm dispersal of

Pseudomonas aeruginosa. J. Bacteriol. 188, 7344–7353 (2006).

40. Schmidt, I., Steenbakkers, P. J. M., op den Camp, H. J. M., Schmidt, K. & Jetten,

M. S. M. Physiologic and Proteomic Evidence for a Role of Nitric Oxide in

Biofilm Formation by Nitrosomonas europaea and Other Ammonia Oxidizers. J.

Bacteriol. 186, 2781–2788 (2004).

41. Hetrick, E., Shin, J. & Stasko, N. Bactericidal efficacy of nitric oxide-releasing

silica nanoparticles. ACS Nano 2, 235–246 (2008).

42. Pankey, G. A. & Sabath, L. D. Clinical relevance of bacteriostatic versus

bactericidal mechanisms of action in the treatment of Gram-positive bacterial

infections. Clin. Infect. Dis. 38, 864–870 (2004).

43. Pamp, S. J., Gjermansen, M., Johansen, H. K. & Tolker-Nielsen, T. Tolerance to

the antimicrobial peptide colistin in Pseudomonas aeruginosa biofilms is linked

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Mol. Microbiol. 68, 223–240 (2008).

94

Chapter 4.

Catalytic generation of nitric

oxide: the use of ferrous ion as a

reducing agent

4.1. Introduction

In the previous chapter, the presence of copper complex (namely CuDTTCT)

together with nitrite and ascorbic acid solution has been shown to be effective in

enhancing nitric oxide generation, which subsequently is able to suppress biofilm

formation and disperse pre-formed biofilm. However, the potential of this system to be

applied is limited due to the requirement to add a reducing agent. Therefore, it would be

highly desirable to utilise a substance that is readily available in water and capable of

reducing copper. For instance, humic substances have been shown to be able to reduce

copper(II) to copper(I) under natural water conditions.1 Despite the widespread presence

of humic substances in water, the presence of organic matters, including humic

substances, is generally unwanted in chlorinated/chloraminated water system due the

possible formation of disinfection by-products. As a result, the potential of inorganic

reducing agent, such as iron, is investigated here.

Chapter 4

95

Iron, as one of the most abundant trace elements in atmospheric waters, usually

undergoes chemical and photochemical reactions resulting in its rapid cycling between

ferrous (Fe2+) and ferric (Fe3+) ions.2,3 Iron that exists as Fe2+ ion has been shown to

reduce copper(II) species to copper(I),4 and this event has been observed in many

systems. For instance, oxidation of Fe2+ ion in seawater condition is enhanced by 0.4

log units in the presence of Cu2+ ion.5 Subsequently, a strong and rapid reduction of

Cu2+ ion to Cu+ ion was observed. Solid iron in the form of green rusts, which are

mixed iron(II)/iron(III) hydroxides, have been found to rapidly reduce aqueous Cu2+ ion

to form solid metallic copper.6 Contact killing of bacteria on solid iron surface was

observed when it is used in conjunction with Cu2+ solution, which was attributed to the

reduction of Cu2+ to form toxic Cu+ by the iron surface.7 Moreover, copper substituted

iron oxide, which is in solid form, was found to be reduced and released into solution

phase in the presence of aqueous Fe2+.8 Therefore, the interaction between copper and

iron can happen either homogeneously (both in solution) or heterogeneously (only one

species in solution form).

In this chapter, the use of Fe2+ ion in the form of ferrous chloride, as an

alternative reducing agent for the reduction of CuDTTCT and the subsequent generation

of nitric oxide (NO) was tested in different solution and pH conditions. The effect non-

equimolar addition of nitrite and Fe2+ on catalytic generation of NO was also studied.

Moreover, the effect of Fe2+ addition in dispersing pre-formed nitrifying biofilms via

NO-mediated dispersal was investigated.

Chapter 4

96

4.2. Experimental methods

4.2.1. Synthesis of copper dibenzo[e,k]-2,3,8,9-tetraphenyl-1,4,7,10-tetraaza-

cyclododeca-1,3,7,9-tetraene complex (CuDTTCT complex)

The synthesis of dibenzo[e,k]-2,3,8,9-tetraphenyl-1,4,7,10-tetraaza-cyclododeca-

1,3,7,9-tetraene (DTTCT), copper complex and its films were performed according to

the method described in the previous chapter (Section 3.2.1).

4.2.2. NO generation measurement with Fe2+ solution as the reducing agent

NO generation from the catalytic system was measured amperometrically.

CuDTTCT films or PVC films were placed at the bottom of a 20 mL glass vials

equipped with stir bars and filled with 10 mL of testing solution, either the nitrifying

bacteria growth medium (ATCC medium 2265, which consists of three different stock

solutions – stock 1 (final composition in the medium mixture): 25 mM (NH4)2SO4, 3

mM KH2PO4, 0.7 mM MgSO4, 0.2 mM CaCl2, 0.01 mM FeSO4, 0.02 mM EDTA, 0.5

µM CuSO4; stock 2: 40 mM KH2PO4, 4 mM NaH2PO4, adjusted to pH 8 by 10 M

NaOH; stock 3: 4 mM Na2CO3) or phosphate buffer (consists of 137 mM NaCl, 2.7 mM

KCl, 10 mM Na2HPO4 and 1.8 mM KH2PO4, prepared using PBS tablet from Sigma).

Growth medium at pH 6 was prepared by adjusting the pH of stock 2 from 4.5 to 5.5 by

10 M NaOH, and mixing it with stock 1 and 3. Concentrated hydrochloric acid was used

to adjust the pH of phosphate buffer from 7.4 to 7, 6.5, and 6. PVC or CuDTTCT films

were placed at the bottom of the vial and measurement probe was placed approximately

1 cm from the film. Sodium nitrite (Ajax Finechem) and iron chloride tetrahydrate

(Sigma Aldrich, ≥99%) were used as the catalytic reactant. At the end of the

Chapter 4

97

measurement, an NO scavenger namely 2-Phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl

3-oxide (PTIO; Alexis Biochemicals) was added to re-establish the baseline.

4.2.3. Fe speciation analysis

The concentration of Fe2+/Fe3+ in the solution was analysed over time via

ferrozine spectroscopy method. Colour reagent ferrozine (Aldrich), reducing agent

hydroxylamine hydrochloric acid (Sigma Aldrich), and ammonium acetate buffer (Ajax)

were prepared as described by Viollier etc.9 Two mL of sample solution was added into

12 well plates (Corning) containing either bare film (PVC film without CuDTTCT) or

CuDTTCT film, followed by addition of equimolar amount of nitrite and Fe2+. Iron

speciation measurement was performed for two hours, with two 100 µL aliquots of the

sample were collected every 3 minutes or 20 minutes. The first aliquots were treated

with ferrozine and represent the concentration of Fe2+, while the second aliquots were

treated with ferrozine, reducing agent and buffer to depict the concentration of total

iron. The absorbance of the Fe2+‒ferrozine complex was measured using UV-Vis

spectrophotometer (Varian Cary 300) at 562 nm.

4.2.4. Biofilm dispersal assay

Biofilm was formed from a mixed inoculum of nitrifying bacteria that is

commercially available for aquarium water purification which comprised of

Nitrosospira multiformis, Nitrospira marina, and Bacillus sp. (Aquasonic Bio-Culture).

One mL of the mixed inoculum was added to 100 mL of the nitrifying bacteria growth

medium and incubated for 3 days in the dark (30 °C, 100 rpm). The three-day-old

Chapter 4

98

cultures were inoculated into fresh medium and 2 mL aliquots (OD = 0.008) were added

in 35 mm culture dishes with a coverglass bottom (internal glass diameter 22 mm,

ProSciTech) with the presence of PVC or CuDTTCT films. All dishes were incubated in

the dark for three days at 30 °C, 100 rpm. One hour before incubation ended, the

supernatants were removed and centrifuged at 10,000 rpm for 15 mins to separate the

planktonic bacteria. During centrifugation, phosphate buffer pH 7.4 (PBS) was added to

the biofilm to prevent the cells from drying. Planktonic bacteria were re-dispersed in

sterile phosphate buffer pH 6 solution followed by the addition of sodium nitrite and

iron(II) to trigger the production of nitric oxide and incubation was continued for one

hour. At the end of the second incubation step, planktonic bacteria were removed. The

dishes that contain the biofilm were washed twice with PBS and stained with 400 µL of

staining solution consists of 3.34 µM of SYTO-9 and 19.97 µM of propidium iodide in

PBS (LIVE/DEAD® BacLightTM Bacterial Viability Kits L-7007, Molecular Probes

Inc.). Green stains denote viable bacteria, while red and yellow stains denote non-viable

bacteria. Twelve images across the glass bottom were acquired and the surface coverage

of the biofilm was analysed using image analysis software (Fiji/ImageJ). Statistical

analysis was performed using one-way ANOVA followed by Dunnett post hoc analysis

on Prism (GraphPad).

4.3. Result and discussion

4.3.1. Effect of testing solution and pH on NO generation from CuDTTCT-nitrite-

Fe2+ mixture

In a system that contains copper(II), nitrite, and a reducing agent, generation of

nitric oxide from nitrite can occur catalytically via copper(II)/copper(I) redox cycling,

Chapter 4

99

as shown in the previous chapter and also supported by literature.10,11 Oplander et al. has

reported the use of Fe2+ as a reducing agent that is able to prompt copper redox cycling,

whereby the reaction between Fe2+ and Cu2+ can form the active Cu+ species and Fe3+,

which subsequently react with nitrite to form NO.12 Therefore, it is proposed that in the

system where CuDTTCT is used, NO generation will occur based on the following

equations:

Cu2+(DTTCT) + Fe2+ Cu+(DTTCT) + Fe3+ (i)

Cu+(DTTCT) + NO2- + H2O ⇌ NO + 2 OH- + Cu2+(DTTCT) (ii)

The amount of NO that can be generated from CuDTTCT-nitrite-Fe2+ system

using nitrifying bacteria growth medium (at initial pH of 8) was measured (Figure 4.1).

Upon the addition of Fe2+ into nitrifying bacteria growth medium that already contained

nitrite and CuDTTCT, no change in signal was observed, indicating that NO generation

could not occur in this condition. However, when the pH of the nitrifying bacteria

growth medium was decreased to 6, a significant increase in NO concentration was

observed after the addition of Fe2+. Note that no pH change was observed in the

conditions tested here. This instant increase in NO concentration is quickly followed by

a rapid decrease. Consequently, only low level of NO could be detected for the

remaining time. An NO scavenger, namely PTIO was then added (the time of addition

was denoted by black arrow; Figure 4.1). The addition of PTIO caused an instant drop

in the observed signal, confirming that the change in signal previously observed was

caused by NO. As stated earlier, NO generation via copper redox cycling is highly

dependent on the formation of the active copper(I) species. Consequently, adequate

presence of Fe2+ species as the reducing agent is vital. The extent of Fe2+ autoxidation is

increased at higher pH,5,13 thus, it is possible that this autoxidation resulted in

Chapter 4

100

diminished availability of Fe2+, subsequently, no NO generation was observed at higher

pH.

Figure 4.1 Amperometric measurement of NO generation from CuDTTCT-nitrite-Fe2+

system. Nitrifying medium pH 8 (black) and pH 6 (green) were used as testing solutions.

0.5 mM of sodium nitrite was added followed by the addition of 0.5 mM of Fe2+ solution.

PTIO was added (time of addition was denoted by a black arrow) to re-establish the

baseline.

The reduced availability of Fe2+ at higher pH and the involvement of Fe2+ in

initiating copper redox cycling is supported by the Fe2+/Fe3+ speciation study. The

amount of Fe2+ available in the absence of copper at time 0, pH 6 is around 0.33 mM

(Figure 4.2c), however, Fe2+ concentration at a similar time is significantly lower for pH

8 (0.17 mM, Figure 4.2a). It should be noted that a 10 seconds time lag generally occurs

between the addition of Fe2+ and the sampling of the first point (time 0). Therefore, the

low concentration of Fe2+ detected at pH 8 could be ascribed to the rapid oxidation of

Fe2+ to Fe3+ in the nitrifying bacteria growth medium-nitrite solution. The change in

Fe2+/Fe3+ concentration over time also supports the above postulation, as Fe2+ could not

be detected after 10 min of measurement at pH 8, while Fe2+ can still be detected up to

0 500 1000 1500-50

0

50

100

150

200

250

300N

O c

on

ce

ntr

atio

n (

nM

)

Time (s)

Growth medium pH 6

Growth medium pH 8

Chapter 4

101

18 min at pH 6. Once all Fe2+ was oxidised, only Fe3+ was detected for the remaining

time. Moreover, the presence of phosphate in the nitrifying bacteria medium could also

aid in the oxidation reaction, where the reaction between Fe2+ with phosphate can form

iron-phosphate species, such as iron-hydroxyphosphate or iron(III)-phosphate

precipitate.14–16 Indeed, a slight decrease in total iron concentration was observed at the

end of measurement (from ~0.5 mM to ~0.3 mM; Figure 4.2a and c), indicating that a

small portion of iron has precipitated out of the solution. The presence of carbonate ions

was also reported to assist iron(II) oxidation,5 and the nitrifying bacteria growth

medium used in this study contains 4 mM of carbonate ions. In addition, nitrifying

growth medium also contains EDTA (0.02 mM), which might complex with Fe2+,

therefore limiting the amount of Fe2+ even further.

Figure 4.2 Change in Fe oxidation state over time in the presence of bare film and nitrite

in nitrifying bacteria growth medium at (a) pH 8 and (c) pH 6; in the presence of

CuDTTCT film and nitrite in nitrifying bacteria medium at (b) pH 8 and (d) pH 6.

Theoretical concentration of nitrite and iron used is 0.5 mM.

(a)

(c) (d)

pH

8p

H 6

0.0

0.1

0.2

0.3

0.4

0.5

Iron c

oncentr

ation (

mM

) Fe(II)

Fe(III)

Total Fe

0 10 20 30 40

0.0

0.1

0.2

0.3

0.4

0.5

Iron c

oncentr

ation (

mM

)

Time (min)0 10 20 30 40

Time (min)

(b)

Without CuDTTCT With CuDTTCT

Chapter 4

102

When CuDTTCT is present, the oxidation of Fe2+ to Fe3+ occurs at a

significantly faster rate, as Fe2+ cannot be detected after 3 min measurement, either at

pH 6 or 8 (Figure 4.2b and d). Therefore, it appears that a reaction between the copper

complex inside the PVC matrix with the Fe2+ ions in the solution occurred. Interestingly,

the interaction between copper and Fe2+ still occur at pH 8 (Figure 4.2b), although this

interaction did not result in NO generation, which seems to be caused by the insufficient

Fe2+ available in this condition, as stated earlier (Figure 4.2a and c).

In order to delay Fe2+ oxidation rate, NO generation and Fe2+/Fe3+ speciation

testing was performed in different solution. Phosphate buffer (PB) was chosen because

of its compatibility with bacteria osmotic pressure. Although PB has been shown to

interact with Fe2+ causing its oxidation,13 the presence of phosphate, which is one of the

main species responsible for autoxidation and precipitation of Fe2+, in PB is

approximately four times lower than in nitrifying bacteria growth medium. Moreover,

carbonate ions and EDTA is not present in PB. Therefore, it was postulated that

oxidation of Fe2+ in PB would occur at a slower rate, which enables a greater amount of

NO to be generated.

The amount of NO that can be generated in PB was first tested at pH 7. In this

condition, the maximum concentration of NO that can be generated was up to 20 nM,

which was observed after ~1000 s (or ~17 min) of measurement (Figure 4.3). At

slightly lower pH of 6.5, a higher amount of NO can be generated over a longer period

of time, as up to 400 nM of NO was detected after ~2000 s (or ~33 min; Figure 4.3). At

the lowest pH tested (pH 6), a significantly higher amount of NO generated was

observed, where up to 4000 nM NO was detected after ~2500 s (or ~42 min) of

Chapter 4

103

measurement (Figure 4.3). Note that only a small pH drop (less than 0.1) was observed

upon the addition of nitrite-Fe2+ into the system at all pH tested.

Figure 4.3 Amperometric measurement of NO generation from CuDTTCT-nitrite-Fe2+

system. Phosphate buffer solution with different pH (from top to bottom: pH 7, pH 6.5

and pH 6) were used as testing solutions. 0.5 mM of sodium nitrite was added followed by

the addition of 0.5 mM of Fe2+ solution. PTIO was added (time of addition was denoted by

a black arrow) to re-establish the baseline.

Comparing the NO generation result obtained from both solutions tested at a

similar pH (pH 6), it was found that NO generation in PB is 13 times higher than in

nitrifying bacteria growth medium. Moreover, NO generation in PB occurred over a

longer period of time, as NO can still be detected even after 6000 s (100 min) of

measurement. These differences in NO generation was ascribed to the higher

0 2000 4000 6000 8000

0

1000

2000

3000

4000

0

100

200

300

400

0

10

20

30

40

Time (s)

PB pH 6

PB pH 6.5

NO

co

nce

ntr

atio

n (

nM

)

PB pH 7

Chapter 4

104

availability of Fe2+ ions in PB compared to nitrifying bacteria growth medium, as

evidenced by the speciation studies described below.

Rapid Fe2+ oxidation at higher pH was also observed when PB was used as the

testing solution (Figure 4.4). For instance, at the highest pH tested (pH 7), Fe2+ cannot

be detected after 40 min of measurement (Figure 4.4a), while at a slightly lower pH (pH

6.5) oxidation was only completed after 100 min of measurement (Figure 4.4c). As

expected, Fe2+ autoxidation was slowest at pH 6, as around 0.1 mM of iron(II) can still

be detected at the end of 120 min measurement (Figure 4.4e). Comparing the result

from PB at pH 6 with the result obtained from the use of nitrifying bacteria growth

medium at similar pH, it was observed that the oxidation of Fe2+ occurred faster in

nitrifying bacteria growth medium than in PB. Therefore, this result suggests that the

presence of complexing agents such as phosphate, carbonate, and EDTA, is a more

prominent factor in facilitating iron oxidation compared to pH.

Chapter 4

105

Figure 4.4 Change in Fe oxidation state over time in the presence of bare film and nitrite

in PB at (a) pH 7, (c) pH 6.5 and (e) pH 6; in the presence of CuDTTCT film and nitrite in

PB at (b) pH 7, (d) pH 6.5 and (f) pH 6. Theoretical concentration of nitrite and iron used

is 0.5 mM.

Similar to the observation in nitrifying bacteria medium, Fe2+ oxidation in PB

solution with the presence of CuDTTCT occurred faster compared to that in the absence

of CuDTTCT (Figure 4.4b, d, f). At pH 7, Fe2+ concentration diminished in the first 20

min of measurement (Figure 4.4b). Even at a lower pH of 6.5 and 6, Fe2+ concentrations

were depleted after 20 to 40 min of reaction (Figure 4.4d and f). Again, these results

confirmed the presence of copper-iron interaction, which was then followed by nitrite

(b)(a)

(c)

pH

7p

H 6

.5p

H 6

(d)

(e) (f)

0.0

0.1

0.2

0.3

0.4

0.5

Iro

n c

on

cen

tra

tio

n (

mM

)

Fe(II)

Fe(III)

Total Fe

0.0

0.1

0.2

0.3

0.4

0.5

Iro

n c

on

cen

tra

tio

n (

mM

)

0 20 40 60 80 100 120

0.0

0.1

0.2

0.3

0.4

0.5

Iro

n c

on

cen

tra

tio

n (

mM

)

Time (min)0 20 40 60 80 100 120

Time (min)

Without CuDTTCT With CuDTTCT

Chapter 4

106

reduction to NO in this system, as shown in NO generation measurement before (Figure

4.3). Interestingly, the reaction between copper(II) species and iron(II) species takes

place regardless of the pH value, which is in agreement with earlier report at a pH lower

than 7.5.5 Moreover, it seems that pH does not affect the reduction reaction rate of

copper, as speciation study showed that the rate of Fe2+ oxidation (around 20 to 40 min)

is similar across all pH range tested.

Although the interaction between CuDTTCT and Fe2+ was shown to happen at

all pH tested and at a similar time scale (Figure 4.4b, d, f), the amount of NO that was

generated from these systems is different (Figure 4.3), with higher NO generated at

lower pH. This therefore suggests that pH is the main factor for NO generation. It was

reported that at low pH, nitrite will form nitrous acid that could decompose and liberate

NO.17 Consequently, it is possible that the NO generated at low pH does not necessarily

originate from the interaction between the active copper(I) species with nitrite, but

could be formed as a result of the decomposition of nitrite.

4.3.2. Effect of nitrite and Fe2+ concentration on NO generation from CuDTTCT-

nitrite-Fe2+ mixture

The effect of different concentration of nitrite and Fe2+ on NO generation was

investigated in PB pH 6 (Figure 4.5). In the absence of CuDTTCT, low concentration of

NO was generated toward the end of measurement (Figure 4.5, black line), which was

possibly induced by the acidic pH of the solution or the slow decomposition of nitrite in

the presence of iron ions.12,17,18 In the presence of similar nitrite-Fe2+ concentration with

CuDTTCT, NO generation occured faster and at higher concentration (up to 200 nM of

NO was measured) compared to the system that contains nitrite-Fe2+ alone. This result

Chapter 4

107

indicates that the generation of NO in the presence of CuDTTCT predominantly occur

through nitrite reduction via copper redox cycling, instead of via acidic decomposition

of nitrite. When nitrite and Fe2+ concentrations were increased, as expected, an enhanced

generation of NO was subsequently observed. The cumulative NO generation over time

is in agreement to the NO release profile, i.e. higher NO generation with an increase in

the concentration of nitrite-Fe2+ added (second Y-axis, Figure 4.5).

The time needed to reach the maximum value differ depends on the

concentration, with higher concentration needs longer time to reach the maximum point

compared to lower concentration. For instance, the maximum value in NO generation

was reached after measurement was performed for ~1100 s for CuDTTCT-0.2 mM

nitrite-iron(II), while the peak for the CuDTTCT-0.1 mM nitrite-iron(II) system was

reached in ~850 s. After the maximum point in was achieved, the signal started to

decay. At this point, PTIO (black arrow, Figure 4.5) was added to re-establish the

baseline.

Chapter 4

108

Figure 4.5 Amperometric measurements of NO generation from CuDTTCT-nitrite-Fe2+

system (first Y-axis) and its corresponding cumulative NO generated (second Y-axis).

After stable baseline was observed, sodium nitrite was added followed by the addition of

equimolar concentration of Fe2+ solution. PTIO (black arrow) was added to re-establish

the baseline.

NO generation was also tested at a fixed nitrite concentration (0.1 mM) but at

different concentration of Fe2+ (Figure 4.6). When 0.1 mM of Fe2+ was added to nitrite-

containing PB pH 6 (red line; Figure 4.6), up to 250 nM of NO can be generated from

the mixture, with the maximum value was reached after measurement for 1000 s.

Interestingly, a further increase in Fe2+ concentration to 0.25 mM (blue line; Figure 4.6)

did not change the highest NO concentration that can be generated (up to 250 nM).

Instead, a shift in the time needed to reach this maximum NO value was observed,

whereby a longer period of 2000-3000 s was needed to reach the maximum.

Consequently, cumulative NO generation was observed to be 1.4 times higher. When

the concentration of Fe2+ added was reduced by half (0.05 mM, purple line; Figure 4.6)

or was ten times lower (0.01 mM, green line; Figure 4.6), only a slight decrease (50 nM)

in NO measurement was detected, with both systems can achieve the maximum value

0 2000 4000 6000

0

500

1000

1500

2000

NO

co

nce

ntr

atio

n (

nM

)

Time (s)

CuDTTCT-0.4 mM nit-Fe

CuDTTCT-0.2 mM nit-Fe

CuDTTCT-0.1 mM nit-Fe

0.1 mM nit-Fe

0

1E6

2E6

3E6

Cu

mu

lative

NO

ge

ne

rate

d (

nM

)

Chapter 4

109

after 1000 s of measurement. It should be noted that NO generation study was also

performed under fixed concentration of Fe2+ and different concentration of nitrite;

however, it was found that no clear trend can be observed, i.e. it behaved similarly as

when equimolar concentration of nitrite-Fe2+ was used.

Figure 4.6 Amperometric measurements of NO generation from CuDTTCT-nitrite-Fe2+

mixture (left) and its corresponding cumulative NO generated (right). Bare film (control,

black lines) or CuDTTCT film were used. After stable baseline was observed, sodium

nitrite (0.1 mM) was added followed by the addition different concentration of Fe2+

solution. After the fast decrease of NO peaks was observed, PTIO (black arrow) was

added to re-establish the baseline.

4.3.3. Biofilm dispersal in the presence of CuDTTCT-nitrite-Fe2+ solution

The effectivity of NO generated from CuDTTCT-nitrite-Fe2+ in dispersing pre-

formed nitrifying biofilm following the three days growth in nitrifying bacteria growth

medium (ATCC medium 2265) was tested. Unlike the methodology used in the

previous chapter, biofilm dispersal was studied in a PB pH 6 solution, which has been

shown to generate the maximum amount of NO. The buffer replacement from nitrifying

bacteria growth medium (approximate pH of 8) to phosphate buffer solution pH 6 do

0 2000 4000 6000

0

100

200

0

100

200

0

100

200

0

100

200

0

100

200

Time (s)

0.25 mM Fe

0.05 mM Fe

0.1 mM Fe

NO

co

nc

en

tra

tio

n (

nM

) 0.01 mM Fe

0.1 mM Fe (Control)

0 2000 4000 6000

0.0

3.0E5

6.0E5

0.0

3.0E5

6.0E5

0.0

3.0E5

6.0E5

0.0

3.0E5

6.0E5

0.0

3.0E5

6.0E5

Time (s)

Cu

mu

lati

ve N

O g

en

era

ted

(n

M)

Chapter 4

110

not cause any premature biofilm dispersal, as surface coverage analysis showed that

biofilms underwent buffer replacement are not significantly different compared to the

(untreated) control (Figure 4.7), which is in agreement with previous report.19

Figure 4.7 (a) Surface coverage analysis of nitrifying biofilm after buffer and pH change.

Both variations was grown in the absence of CuDTTCT. All values shown are normalised

to the surface coverage of control. Error bars indicate standard error between replicates

(n = 2). * p ≤ 0.05 compared to the control. Confocal laser scanning microscopy images of

nitrifying bacteria biofilm (b) untreated (control), (c) after buffer replacement with

phosphate buffer pH 6. Scale bar = 100 µm.

The addition of CuDTTCT was found not to affect biofilm growth, as the

surface coverage of biofilm that was grown in the presence of CuDTTCT is similar with

the one grown in the absence of CuDTTCT (control, Figure 4.8a-c). When the pre-

formed biofilms were treated with 0.1 mM and 0.2 mM of nitrite and Fe2+, a ~35%

reduction in biofilm surface coverage compared to the control was observed (Figure

4.8a, d-e). Enhanced biofilm surface coverage reduction was observed when 0.4 mM of

nitrite-Fe2+ was added into the wells, which resulted in a 45% reduction in biofilm

surface coverage compared to the control (Figure 4.8f). Although the addition of iron

species has been shown to disperse pre-formed Pseudomonas aeruginosa biofilm, the

effect was not observed here as the biofilms treated with Fe2+ alone at varying

(a) (b)

(c)

Control

PB pH 6

Control PB pH 60.0

0.5

1.0

1.5

Re

lative

su

rfa

ce

co

ve

rag

e

Chapter 4

111

concentrations (0.1 mM, 0.2 mM, and 0.4 mM) did not result in significant changes in

cells surface coverage compared to control (Figure 4.9). Moreover, the addition of

nitrite was also unable to trigger biofilm dispersal for nitrifying bacteria, as reported

before.19 Therefore, all three components (CuDTTCT, nitrite, and Fe2+) must be present

together in order to achieve biofilm dispersal, which suggests the involvement of NO

that can be generated in the presence of these components (Figure 4.5).

Figure 4.8 (a) Surface coverage analysis of nitrifying bacteria biofilm after addition of

nitrite and Fe2+ in PB pH 6. All values shown are normalised to the surface coverage of the

control (PVC film). Error bars indicate standard error between replicates (n = 3). *p ≤

0.05 compared to the control and 0 mM variation. Confocal laser scanning microscopy

images of nitrifying bacteria biofilm (b) grown in the presence of PVC film (control), (c)

grown in the presence of CuDTTCT film (0 mM) and (d) treated with 0.1 mM nitrite-Fe2+,

(e) treated with 0.2 mM nitrite-Fe2+, and (f) treated with 0.4 mM nitrite-Fe2+. Scale bar =

100 µm.

0.0

0.5

1.0

1.5

**

Re

lative

su

rfa

ce

co

ve

rag

e

Nitrite-iron concentration (mM)Control 0 0.1 0.2 0.4

*

(b)

(c)

(d)

(e)

(f)

Control

0 mM

0.1 mM

0.2 mM

0.4 mM

(a)

Chapter 4

112

Figure 4.9 (a) Surface coverage analysis of nitrifying biofilm after the addition of Fe2+ in

PB pH 6. All values shown are normalised to the surface coverage of the control. Error

bars indicate standard error between replicates (n = 2). *p ≤ 0.05 compared to the control.

Confocal laser scanning microscopy images of nitrifying biofilm (b) untreated (control),

(c) treated with 0.1 mM Fe2+, (d) treated with 0.2 mM Fe2+, and (e) treated with 0.4 mM

Fe2+. Scale bar = 100 µm.

The activity of NO to disperse pre-established biofilm is known for various

bacteria species, including nitrifying bacteria.19–22 NO could affect the switch between

biofilm and planktonic phase through alteration in the secondary cellular messenger

level, i.e. cyclic di-GMP level. Because NO-mediated dispersal occurs via a signalling

pathway, cell viability will still be maintained after this treatment, which is confirmed

by the observation presented in the CLSM analysis (Figure 4.8b-f). Moreover, NO is

also known to cause reduction in cell aggregation.23 Indeed, confocal images showed

that in systems without nitrite-Fe2+ treatment, both in the absence or presence of

CuDTTCT, significant cells aggregation could be seen (Figure 4.8a-b). However, when

the biofilm was treated with nitrite and Fe2+, reduction in cells aggregation was

observed (Figure 4.8d-f).

Comparing the result obtained in the previous chapter (section 3.3.4) and

presented herein, it can be concluded that NO generated catalytically via copper redox

(a)

(c) 0.1 mM

(b) Control (d) 0.2 mM

0.4 mM(e)

0.0

0.5

1.0

1.5

Re

lative

su

rfa

ce

co

ve

rag

e

Iron concentration (mM)

Control 0.1 0.2 0.4

Chapter 4

113

cycling is effective in dispersing pre-formed biofilm, regardless of the reducing agent

used. Moreover, NO-mediated dispersal was also observed under different solution

condition, i.e. nitrifying bacteria growth medium at pH 8 and PB pH 6, which implies

that biofilm dispersal by NO is not dependent on the buffer condition, and interestingly,

achievable in an environment that does not support growth such as PB.

4.4. Summary

Herein, Fe2+ species was studied as an alternative reducing agent for catalytic

generation of NO from nitrite in the presence of CuDTTCT catalyst. Amperometric

measurement showed that the combination of CuDTTCT-nitrite-Fe2+ was only able to

generate NO in nitrifying bacteria growth medium at pH 6 and not at pH 8, due to the

rapid autoxidation and precipitation of Fe2+ at high pH. Moreover, iron speciation study

showed that an interaction between CuDTTCT and Fe2+ exist at all pH conditions. The

oxidation rate of Fe2+ was faster in the presence of CuDTTCT compared to control and

at a similar kinetics for all pH conditions studied. Consequently, this interaction resulted

in the reduction of copper(II) species to copper(I), which can catalyse nitrite reduction

to form NO. Similar observation was found in PB systems, whereby higher NO was

generated at lower pH. Moreover, it was also found that the presence of complexing

agents, particularly phosphate, carbonate, and EDTA, is a more prominent factor in

influencing Fe2+ autoxidation compared to pH. Consequently, PB pH 6 was found to

generate the highest amount NO from all the different buffer medium and pH conditions

tested here. The potential of this system in dispersing pre-formed nitrifying biofilm was

performed in PB at pH 6. The NO generated in this condition is effective at dispersing

biofilm and reducing cell aggregations. The results from this and previous chapter

Chapter 4

114

therefore suggest that NO-mediated biofilm dispersal is not affected by the choice of

reducing agent or by the buffer condition.

4.5. References

1. Pham, A., Rose, A. & Waite, T. Kinetics of Cu (II) reduction by natural organic

matter. J. Phys. Chem. A 116, 6590–6599 (2012).

2. Sedlak, D. L. & Hoigné, J. The role of copper and oxalate in the redox cycling of

iron in atmospheric waters. Atmos. Environ. Part A. Gen. Top. 27, 2173–2185

(1993).

3. Willey, J. D. & Whitehead, R. F. Oxidation of Fe (II) in Rainwater. Environ. Sci.

Technol. 39, 2579–2585 (2005).

4. Matocha, C. J., Karathanasis, A. D., Rakshit, S. & Wagner, K. M. Reduction of

copper(II) by iron(II). J. Environ. Qual. 34, 1539–1546 (2005).

5. González, A. G., Pérez-Almeida, N., Magdalena Santana-Casiano, J., Millero, F.

J. & González-Dávila, M. Redox interactions of Fe and Cu in seawater. Mar.

Chem. 179, 12–22 (2016).

6. O’Loughlin, E. J., Kelly, S. D., Kemner, K. M., Csencsits, R. & Cook, R. E.

Reduction of AgI, AuIII, CuII, and Hg II by FeII/FeIII hydroxysulfate green rust.

Chemosphere 53, 437–446 (2003).

7. Mathews, S., Kumar, R. & Solioz, M. Copper reduction and contact killing of

bacteria by iron surfaces. Appl. Environ. Microbiol. 81, 6399–6403 (2015).

8. Frierdich, A. J. & Catalano, J. G. Fe(II)-mediated reduction and repartitioning of

structurally incorporated Cu, Co, and Mn in iron oxides. Environ. Sci. Technol.

46, 11070–11077 (2012).

9. Viollier, E., Inglett, P. W., Hunter, K., Roychoudhury, A. N. & Van Cappellen,

P. The ferrozine method revisited: Fe(II)/Fe(III) determination in natural waters.

Chapter 4

115

Appl. Geochemistry 15, 785–790 (2000).

10. Opländer, C. et al. Characterization of novel nitrite-based nitric oxide generating

delivery systems for topical dermal application. Nitric Oxide 28, 24–32 (2013).

11. Oh, B. K. & Meyerhoff, M. E. Catalytic generation of nitric oxide from nitrite at

the interface of polymeric films doped with lipophilic Cu(II)-complex: a potential

route to the preparation of thromboresistant coatings. Biomaterials 25, 283–293

(2004).

12. Opländer, C. et al. Redox-mediated mechanisms and biological responses of

copper-catalyzed reduction of the nitrite ion in vitro. Nitric Oxide 35, 152–164

(2013).

13. Welch, K. D., Davis, T. Z. & Aust, S. D. Iron autoxidation and free radical

generation: effects of buffers, ligands, and chelators. Arch. Biochem. Biophys.

397, 360–369 (2002).

14. Van Der Grift, B., Rozemeijer, J. C., Griffioen, J. & Van Der Velde, Y. Iron

oxidation kinetics and phosphate immobilization along the flow-path from

groundwater into surface water. Hydrol. Earth Syst. Sci. 18, 4687–4702 (2014).

15. Grift, B. van der et al. Fe hydroxyphosphate precipitation and Fe ( II ) oxidation

kinetics upon aeration of Fe ( II ) and phosphate-containing synthetic and natural

solutions. Geochim. Cosmochim. Acta 186, 71–90 (2016).

16. Baken, S., Moens, C., van der Grift, B. & Smolders, E. Phosphate binding by

natural iron-rich colloids in streams. Water Res. 98, 326–333 (2016).

17. Lundberg, J. O., Weitzberg, E. & Gladwin, M. T. The nitrate-nitrite-nitric oxide

pathway in physiology and therapeutics. Nat. Rev. Drug Discov. 7, 156–167

(2008).

18. Van Cleemput, O. & Baert, L. Nitrite stability influenced by iron compounds.

Soil Biol. Biochem. 15, 137–140 (1983).

19. Schmidt, I., Steenbakkers, P. J. M., op den Camp, H. J. M., Schmidt, K. & Jetten,

M. S. M. Physiologic and Proteomic Evidence for a Role of Nitric Oxide in

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Biofilm Formation by Nitrosomonas europaea and Other Ammonia Oxidizers. J.

Bacteriol. 186, 2781–2788 (2004).

20. McDougald, D., Rice, S. A., Barraud, N., Steinberg, P. D. & Kjelleberg, S.

Should we stay or should we go: mechanisms and ecological consequences for

biofilm dispersal. Nat. Rev. Microbiol. 10, 39–50 (2011).

21. Craven, M. et al. Nitric Oxide-Releasing Polyacrylonitrile Disperses Biofilms

Formed by Wound-Relevant Pathogenic Bacteria. J. Appl. Microbiol. 120, 1085–

1099 (2016).

22. Barnes, R. J. et al. Nitric oxide treatment for the control of reverse osmosis

membrane biofouling. Appl. Environ. Microbiol. 81, 2515–2524 (2015).

23. Schlag, S., Nerz, C., Birkenstock, T. A., Altenberend, F. & Götz, F. Inhibition of

staphylococcal biofilm formation by nitrite. J. Bacteriol. 189, 7911–7919 (2007).

117

Chapter 5.

Iron complex facilitated copper

redox cycling for nitric oxide

generation

(This chapter is based on the work published in ACS Applied Materials and Interfaces

volume 8, issue 44, page 30502-30510, 2016.)

5.1. Introduction

The studies presented in the previous chapters (Chapter 3 and 4) have shown that

aqueous reducing agents are required for nitric oxide (NO) generation. However, these

reducing agents are not always readily available in water. The need for continuous

addition of an external reducing agent is not desirable as it is costly, difficult to control

the dose and the added reducing agent might affect water composition and quality.

Moreover, the presence of oxidants chlorine and chloramine, which are commonly

added to water systems as disinfectants, might neutralise the reducing agent.

Consequently, a reducing agent that can be immobilised together with the copper

catalyst within a poly(vinyl chloride) (PVC) matrix will be highly beneficial. In

particular, the utilisation of iron complex is of interest.

Chapter 5

118

Iron species, which is capable of reducing copper catalyst as shown in Chapter 4,

is known to assist various redox processes. For instance, an iron-humic acid complex

was found to accelerate the microbial reduction of iron(III) oxide.1 Several parameters

should be considered in ligand selection suitable for complex formation. For instance,

iron ligands can be either oxygen, nitrogen, or sulphur atoms-based. Oxygen-based

ligands, such as citrate, prefer to complex with iron(III), consequently, these ligands

will promote the oxidation of iron(II) to iron(III) and a decrease in iron reduction

potential can be observed.2 On the other hand, nitrogen- and sulphur-based ligands can

stabilise iron(II), therefore they can inhibit iron(II) oxidation and cause an increase in

iron reduction potential.2,3 Therefore, nitrogen- or sulphur-based ligands are preferred

in this study, due to the required presence of iron(II) species in facilitating

copper(II)/copper(I) redox cycling for NO generation. Moreover, the ligand should also

able to accommodate the change in metal ion size from reduction/oxidation process that

will occur.4 DTTCT (dibenzo[e,k]-2,3,8,9-tetraphenyl-1,4,7,10-tetraaza-cyclododeca-

1,3,7,9-tetraene), which is used as the ligand for the copper complex catalyst employed

in this study, is a nitrogen-based ligand that has been proven capable of accommodating

metal ion size change, as evidenced by x-ray photoelectron spectroscopy (XPS) study

presented in Chapter 3. Interestingly, it was reported that ligands which prefer

copper(II) species will possess an affinity for other bivalent metals, such as iron(II).3

Therefore, DTTCT seems to be a suitable ligand for iron complexation in this study.

Herein, iron and copper complexes (FeDTTCT and CuDTTCT, respectively)

were immobilised within a PVC matrix. The ability of the immobilised FeDTTCT in

facilitating the reduction of copper(II) in CuDTTCT to the active copper(I) species and

its subsequent generation of NO via nitrite reduction was assessed. The effectiveness of

Chapter 5

119

the mixed metal system in inhibiting nitrifying bacteria biofilm formation without any

additional reducing agent was also examined.

Scheme 5.1 DTTCT structure

5.2. Experimental methods

5.2.1. Synthesis of dibenzo[e,k]-2,3,8,9-tetraphenyl-1,4,7,10-tetraaza-cyclododeca-

1,3,7,9-tetraene ligand and the metal complexes

The synthesis of dibenzo[e,k]-2,3,8,9-tetraphenyl-1,4,7,10-tetraaza-cyclododeca-

1,3,7,9-tetraene (DTTCT) and copper complex were performed according to the method

described in the previous chapter (Section 3.2.1 and Section 4.2.1). Synthesis of iron

complex was carried out by mixing iron(III) chloride hexahydrate (0.05 mol; Ajax APS)

with DTTCT (0.01 mol) in ethanol and refluxed at 85 °C for 6 hours. After cooling

overnight, the resultant precipitate was separated by centrifugation, washed with cold

ethanol and dried in vacuum desiccator yielding a dark brown powder.

The films were synthesised by mixing the metal complex with pre-dissolved

poly(vinyl chloride) (PVC) in tetrahydrofuran (THF, Chem-Supply) solution (0.3 mL,

66 mg/L) in an ultrasonic bath. Round glass cover slips (18 mm diameter, ProSciTech)

Chapter 5

120

were washed with dilute nitric acid, acetone, and ethanol followed by overnight drying

at 110 °C before being used as the cast template. Bare film (PVC without any metal

complexes, typical weight ~20 mg) was used as the control. To prepare the films that

contain a mixture of the CuDTTCT and FeDTTCT metal complexes, CuDTTCT (0.25

mg, 0.5 mg, 0.75 mg, and 1 mg; corresponding to 1.25 wt%, 2.5 wt%, 3.5 wt%, and 5

wt%, respectively) and FeDTTCT (equal loading) were mixed with the pre-dissolved

PVC in THF solution in an ultrasonic bath, and then casted on the round glass cover

slips. All films were dried at 50 °C for 12 hours and peeled off from the glass cover

slips.

5.2.2. Characterisation of metal complexes

Analysis of the structure was performed in deuterated dimethyl sulfoxide solvent

by 1H-NMR spectroscopy using a Bruker Avance III HD 400 MHz. Thermal analysis of

the ligands and metal complexes were carried out by Differential Scanning Calorimetry

(DSC; Perkin Elmer) to confirm the metal complex formation. Around 3 to 6 mg of the

sample were placed in a closed aluminium pan, with empty closed aluminium pan as the

reference. The temperature was increased at a rate of 10 °C/mins under nitrogen

environment (50 mL/min). Results shown are a representative thermogram obtained

from duplicates.

A field emission scanning electron microscopy (SEM; FEI Nova NanoSEM 230)

equipped with an energy dispersive X-ray detector (EDS; Bruker Silicon Drift Energy

Dispersive X-ray detector) was used to show the immobilisation of copper and iron

complexes in PVC matrix. Prior to imaging, the samples were attached to 10 mm metal

Chapter 5

121

mounts by carbon tape and sputter coated with chromium. Data analysis was performed

with Esprit EDS software.

XPS measurements were performed using an ESCALAB 220iXL spectrometer

with monochromated Al Kα (energy 1486.6 eV). Film samples were cleaned by brief

immersion in toluene prior to analysis. Reaction was performed by immersing the pre-

cleaned sample in a 10 mM phosphate buffer solution pH 6 (Sigma, pH adjusted by

concentrated hydrochloric acid) containing 1 mM sodium nitrite (Ajax Finechem) for 10

min. All film samples were dried in a vacuum desiccator for a minimum of 2 days

before analysis. A random spot on the film with diameter around 0.5 mm was chosen

for the analysis. All spectra were calibrated to the C1s peak signal at 284.8 eV.

5.2.3. NO generation measurement

NO generation from the system was measured amperometrically by using an

Apollo TBR4100 Free Radical Analyser (World Precision Instrument) equipped with

ISO-NOP 2 mm probe. Calibration was performed using S-nitroso-N-

acetylpenicillamine (SNAP; Sigma) and copper sulphate solution according to

manufacturer’s protocol. The test film was placed at the bottom of a 20 mL glass vial

equipped with stir bars and filled with 10 mL of 10 mM phosphate buffer solution pH 6.

After a stable baseline was observed, 1 mM sodium nitrite was added to the glass vial.

At the end of the measurement, an NO scavenger namely 2-phenyl-4,4,5,5-

tetramethylimidazoline-1-oxyl 3-oxide (PTIO; Alexis Biochemicals) was added to re-

establish the baseline. All experiments were performed in the presence of ambient

oxygen. Result shown are a representative graph obtained from a minimum of three

replicates.

Chapter 5

122

5.2.4. Biofilm assay

The effect of the mixed metal complex films on nitrifying biofilms was assessed

as previously described (Section 3.2.4). Biofilm was formed from a mixed inoculum of

nitrifying bacteria that is available commercially for the purification of aquarium water

and is comprised of ammonia-oxidising bacteria Nitrosospira multiformis, nitrite-

oxidising bacteria Nitrospira marina, and heterotrophic bacteria Bacillus sp. (Aquasonic

Bio-Culture). One millilitre of the mixed inoculum was added into 100 mL of the

nitrifying growth medium (ATCC medium 2265 consisting of three different stock

solutions – stock 1 (final composition in the medium mixture): 25 mM (NH4)2SO4, 3

mM KH2PO4, 0.7 mM MgSO4, 0.2 mM CaCl2, 0.01 mM FeSO4, 0.02 mM EDTA, 0.5

µM CuSO4; stock 2: 40 mM KH2PO4, 4 mM NaH2PO4, adjusted to pH 8 by 10 M

NaOH; stock 3: 4 mM Na2CO3) and incubated for 3 days in the dark (30 °C, 100 rpm).

The three-day-old cultures were inoculated into fresh medium and 2 mL aliquots (OD =

0.008) were added into 12-well plates with the presence of a bare film (control) or a

mixed metal film. The plates were then incubated in the dark for 3 days at 30 °C, 100

rpm. At the end of incubation, the bacteria supernatants were removed and centrifuged

at 12,000 rpm for 15 min and washed in phosphate buffer saline (PBS; Sigma) once to

separate planktonic bacteria. The supernatants were filtered through a 0.2 µm membrane

and used to measure the extent of copper and iron leaching from the films by ICP-OES

(PerkinElmer OPTIMA 7300). The biofilms were washed twice to remove the loosely

attached cells. The biomass was measured by quantifying total protein content by using

the bicinchoninic acid method (BCA assay; Sigma). BCA working reagent (2 mL) was

added into each of the biofilm and planktonic cells, followed by incubation for 30 mins

at 37 °C, 100 rpm. A preliminary experiment has shown that the presence of a mixed

Chapter 5

123

metal film did not interfere with the assay, even after the incubation step. Standard

solutions as per manufacturer’s instructions were used for each experiment.

Surface coverage analysis and cell viability assessment were performed using

confocal laser scanning microscopy (Olympus FluoViewTM FV1000) of LIVE/DEAD-

stained biofilm cells. For microscopic observation, an aliquot of the nitrifying culture

was added into a 35 mm culture dishes with coverglass bottom (internal glass diameter

22 mm, ProSciTech) with the presence of a bare film (control) or a mixed metal film,

followed by incubation in the dark for three days at 30 °C, 100 rpm. At the end of

incubation, the supernatants were removed and biofilms attached on the glass

substratum were washed twice with PBS. Adhered cells were stained with SYTO-9 and

propidium iodide according to manufacturer’s instruction (LIVE/DEAD BacLightTM

bacterial viability kits L-7007, Molecular Probes Inc.) and incubated at room

temperature for a minimum of 15 min. Twelve pictures across the glass bottom were

obtained and surface coverage analysis was performed on live cells (green channel)

using image analysis software (Fiji/ImageJ). All statistical analyses were performed on

GraphPad Prism 6 (GraphPad Software) using one-way ANOVA method followed by

Dunnett’s posthoc analysis.

5.3. Result and discussion

5.3.1. Confirmation of metal complexes formation

Copper complex (CuDTTCT) and iron complex (FeDTTCT) were synthesised

and characterised. 1H-NMR analysis of the metal complexes shows similar spectra as

the one observed for DTTCT, thus confirming the presence of DTTCT in the metal

Chapter 5

124

complex (Figure 3.1). Thermal analysis by DSC shows ligand DTTCT only exhibited

one strong endothermic peak at 124 °C. Further complexation with copper generated

four endothermic peaks at 121 °C, 156 °C, 204 °C and 269 °C (Figure 5.1). The

sharpest peak of 269 °C could be ascribed to the melting point-decomposition of

CuDTTCT sample. This shift in melting point from 124 °C for DTTCT to 269 °C

confirmed the successful formation of the complex. For FeDTTCT sample, one

exothermic peak at 153 °C was observed, followed by 3 endothermic peaks at 165 °C,

193 °C, and 248 °C. The exothermic peak at 153 °C is possibly due to the

decomposition of the sample, as observed previously by Breviglieri et al.5 The absence

of sharp endothermic peak at 124 °C again confirmed the formation of metal complex.5–

7 Finally, SEM-EDS mapping was performed on the PVC film that contains both

CuDTTCT and FeDTTCT (mixed metal film). Copper was uniformly detected

throughout the samples, while iron mapping showed aggregated distribution (Figure

5.2). Visual observation of the films also confirms this as dark brown aggregates (the

iron complex) were observed on the surface of the samples. These results confirmed the

presence of copper and iron in the film, and subsequently, in the complexes.

Figure 5.1 DSC thermograms of DTTCT, CuDTTCT, and FeDTTCT with a heating rate

of 10 °C/min.

0 100 200 300-12

-8

-4

0

4

8

Heat flo

w (

mW

)

Temperature (°C)

DTTCT

0 100 200 300

Temperature (°C)

CuDTTCT

0 100 200 300

Temperature (°C)

FeDTTCT

Chapter 5

125

Figure 5.2 (a) SEM images of mixed metal film (CuDTTCT+FeDTTCT) and its

corresponding elemental mapping of (b) copper and (c) iron. (d) EDS spectra of the mixed

metal film. Other peak labels are removed for clarity.

5.3.2. Nitric oxide generation from CuDTTCT+FeDTTCT films in the presence of

nitrite

Evaluation of NO release in systems containing mixed CuDTTCT+FeDTTCT

metal complexes films (along with their associated set of controls) was performed

amperometrically, where NO peak can be observed upon the addition of 1 mM nitrite

into the systems (Figure 5.3). Low and stable levels of NO was generated in the various

control systems. In the case of the bare PVC film (without metal complex) and film with

1 mg FeDTTCT loading, up to 25 nM NO were generated, which is thought to result

from the formation of nitrous acid from nitrite and its subsequent decomposition in

acidic pH.8 A higher level of 50 nM NO was generated in the presence of film with 1

mg CuDTTCT loading. The interactions of copper(II) with various NO donors, such as

(a) (b) (c)

(d)

Chapter 5

126

nitrite and S-nitrosothiols, have been reported to generate low levels of NO.9–11

Interestingly, when the combination of CuDTTCT+FeDTTCT mixed metal film at

(0.5+0.5) mg loading was used, a surge of up to 125 nM NO was generated in the first

~750 s of measurement, followed by a slow decrease in NO concentration. The addition

of the NO scavenger PTIO at the end of the measurement caused a sudden decrease in

the signal, confirming the generation of NO. The significant generation of NO from

nitrite in the presence of the mixed metal complexes is most likely facilitated by

copper(II)/copper(I) redox cycling, with iron complex acting as reducing agent, as

suggested by the following equations:

Cu2+(DTTCT) + Fe2+(DTTCT) ⇌ Cu+(DTTCT) + Fe3+(DTTCT) (i)

Cu+(DTTCT) + NO2– + H2O ⇌ NO + 2 OH‒ + Cu2+(DTTCT) (ii)

As shown in previous chapters, NO generation from nitrite can occur via

copper(II)/copper(I) redox cycling in the presence of a reducing agent (such as ascorbic

acid and Fe2+ ions). Copper(I) species, which was formed from reduction of copper(II)

species by the reducing agent, is the most active species for NO production from

nitrite.12 Copper(II) reduction by iron species and importantly, the generation of NO

from nitrite via reaction with the formed copper(I) species, have been observed by

several studies,9,13–17 including the one shown in Chapter 4. However, up to this stage,

copper(II) reduction by iron(II) has been reported in solution form, either

homogeneously (both metals as dissolved species) or heterogeneously (with one metal

in solution form).16 Eventually, when the formed copper(I) is oxidised to copper(II) and

iron-facilitated reduction of copper(II) does not happen anymore, NO generation will

stop.

Chapter 5

127

Doubling the mixed metal complexes loading to (1+1) mg caused an up to 2-

fold increase in NO generation in the presence of nitrite, which was detected within

similar time frame as that of the lower loading of metal complexes (in the first 750 s).

This was again followed by a similar slow decrease in NO concentration prior to PTIO

addition. This enhanced NO generation was due to the presence of more catalytic active

sites with the increasing amount of metal complexes.

Figure 5.3 Amperometric measurements of NO generation from mixed metal films

containing a mixture of metal complex (CuDTTCT+FeDTTCT; 1+1 and 0.5+0.5) with the

addition of 1 mM nitrite. Three different controls were used (bare film, 1 mg CuDTTCT

film, 1 mg FeDTTCT film) in the presence of 1 mM nitrite solution. PTIO (100 µM; black

arrow) was added at the end of measurement to re-establish the baseline.

The iron complex-facilitated copper(II)/copper(I) redox cycling is supported by

the observed changes in the binding energy of iron and copper species, as revealed by

XPS analysis of the mixed metal complexes before and after addition of nitrite (Figure

5.4). The XPS spectra of FeDTTCT in the mixed metal film can be deconvoluted into

two Fe 2p3/2 peaks at 711.3 eV and 713.9 eV, with a satellite peak at 719.4 eV (Figure

5.4b, before reaction), which is similar to that of the as-synthesised FeDTTCT (Figure

0 1000 2000 3000 4000

-50

0

50

100

150

200

250

NO

co

nce

ntr

atio

n (

nM

)

Time (s)

(1+1) mg

(0.5+0.5) mg

Cu 1 mg

Fe 1 mg

bare

Chapter 5

128

5.4a). Note that there is currently no published data on the XPS spectra of FeDTTCT,

and the position of iron(II) and iron(III) peaks in Fe 2p region could differ depending on

the ligand species.18 The CuDTTCT in the mixed metal film mainly exists as copper(II)

species, with detection of Cu 2p3/2 peak at 934.7 eV along with two satellite peaks at

940.1 eV and 943.7 eV (Figure 5.4c, before reaction). Following NO generation in the

presence of nitrite, a significant Fe 2p3/2 peak shift from 711.3 eV to 712.1 eV was

observed, which was followed by the shift in the corresponding satellite peak to 718.6

eV and the disappearance of the second Fe 2p3/2 peak (Figure 5.4b, after reaction). This

shift of Fe 2p3/2 peak to higher binding energy indicates oxidation of the iron

complex.18,19 Post reaction XPS analysis also revealed the emergence of copper(I) peak

at 933.9 eV (Figure 5.4c, after reaction), which as stated previously, is the active species

in NO generation reaction. Therefore, this observation implies the role of the iron

complex as a reducing agent, facilitating the catalytic activity of copper through

copper(II)/copper(I) redox cycling for generation NO from nitrite. Further studies on

catalytic NO generation in solutions containing nitrite and ascorbic acid (as reducing

agent) detected a significantly higher NO concentration in the presence of CuDTTCT

than that of FeDTTCT (Figure 5.5). This further validates the role of FeDTTCT in

facilitating the reduction of copper(II) to copper(I) in the mixed metal systems, rather

than catalysing the reduction of nitrite to NO.

Chapter 5

129

Figure 5.4 XPS spectra of Fe 2p region of (a) as synthesised FeDTTCT, (b)

CuDTTCT+FeDTTCT (mixed) film before and after reaction. (c) XPS spectra of Cu 2p

region of CuDTTCT+FeDTTCT (mixed) film before and after reaction. Reaction was

performed in the presence of 1 mM of nitrite for 10 mins.

740 730 720 710 700

Counts

(s

-1)

Binding energy (eV)

Before reaction

After reaction

970 960 950 940 930 920

Cu2+

After reaction

Binding energy (eV)

Cu+

Cu2+

Before reaction

(b) (c)

(a)

740 730 720 710 700

Co

un

ts (

s-1)

Binding energy (eV)

FeDTTCT as synthesised

Chapter 5

130

Figure 5.5 Amperometric measurements of NO generation from CuDTTCT film (1 mg

loading) or FeDTTCT film (1 mg loading) in the presence of 1 mM nitrite and 1 mM

ascorbic acid. Bare film (PVC film without the addition of metal complex) was used as the

control. Black arrow denotes the addition of ascorbic acid.

5.3.3. Antibiofilm activity of CuDTTCT+FeDTTCT mixed metal film by the

involvement of catalytically generated NO

The potential of CuDTTCT+FeDTTCT mixed metal complexes as NO-

generating material for antibiofilm application was investigated against the

ubiquitously-occurring nitrifying bacterial biofilms. Nitrifying bacteria of the

Nitrosospira genera are commonly known as ammonia oxidising bacteria or AOB,

capable of oxidising ammonia to nitrite. Nitrite production from ammonia occurs via a

two-step process involving the conversion of ammonia to hydroxylamine catalysed by

the enzyme ammonia monooxygenase (Amo), which then followed by the conversion of

hydroxylamine to nitrite by the enzyme hydroxylamine oxidoreductase (Hao). A mixed

inoculum containing nitrifying bacteria was used to grow nitrifying biofilms for three

days in a specific ammonia oxidising bacteria culture medium in the presence of bare

0 1000 2000 3000 4000 5000 6000

0

1000

2000

3000

4000

5000

NO

concentr

ation (

nM

)

Time (s)

CuDTTCT

Bare

FeDTTCT

Chapter 5

131

(PVC-only) film, whereby 16 mM nitrite was detected in the system (by ion

chromatography) after 3 days. Low level of 0.1 mM nitrate was also detected in the

system, potentially due to the presence of nitrite oxidising bacteria (NOB) Nitrospira

spp. in the inoculum, which are capable of oxidising nitrite to nitrate. The biofilm

growth was also characterised by detection of ~300 µg/mL protein on the well and bare

film surfaces, while ~100 µg/mL of protein was detected in the suspended planktonic

phase. In this self-produced nitrite environment, the mixed metal systems exhibited a

unique anti-biofilm effect, as described in the following.

The extent of biofilm and planktonic growth in the presence of increasing mixed

metal loading of (0.25 mg Cu + 0.25 mg Fe), (0.5 mg Cu + 0.5 mg Fe) and (0.75 mg Cu

+ 0.75 mg Fe) along with their corresponding copper and iron leached ions are

presented in Figure 5.6 and Table 5.1, respectively. The presence of (0.25+0.25) mg

mixed metal complexes did not affect bacterial growth, as comparable biofilm and

planktonic biomass as those detected in the control was observed (Figure 5.6a).

Increasing the metal loading to (0.5+0.5) mg caused a 20% reduction in biofilm biomass

relative to the control growth and correspondingly, a 20% increase in planktonic

biomass. Ultimately, at (0.75+0.75) mg metal loading, a 36% decrease in biofilm

biomass was observed, which was followed by the detection of a 70% increase in

planktonic biomass. It should be noted that a comparable total leaching of ~0.5 mg/L of

Cu and Fe was detected at all mixed metal loading (Table 5.1). Surface coverage

analysis by confocal laser scanning microscopy (CLSM) (Figure 5.6b) further

confirmed the abovementioned trend from the protein assay, with a 56% overall

reduction in biofilm surface coverage observed for the (0.75+0.75) mg mixed metal

system relative to the control. Note that the CLSM analysis was carried out to account

for potential interference of biofilm EPS in the protein assay.

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132

Figure 5.6 (a) Protein measurements of nitrifying bacteria biomass grown in the presence

of various loading of mixed metal film. All values shown are normalised to the bacteria

biomass grown in the presence of bare film (control, inset). Error bars indicate standard

error between replicates (n = 4); *p ≤ 0.05 against the control. (b) Surface coverage

analysis of nitrifying biofilm. All values shown are normalised to the surface coverage of

control. Error bars indicate standard error between replicates (n = 3); *p ≤ 0.05 against

the control. Confocal laser scanning images of nitrifying biofilm grown in the presence of

(c) bare film, (d) mixed metal film with (0.25+0.25) mg loading, (e) (0.5+0.5) mg loading,

and (f) (0.75+0.75) mg loading.

(a)

(e)

(f)

(b)

(c)

(d)

0.0

0.5

1.0

1.5

2.0*

*

Rela

tive p

rote

in c

oncentr

ation

CuDTTCT+FeDTTCT loading (mg)

Biofilm

Planktonic

*

Control (0.25+0.25) (0.5+0.5) (0.75+0.75)

0

100

200

300

Pro

tein

co

nc

en

tra

tio

n

(µg

/mL

)

Control

0.0

0.5

1.0

1.5

2.0

Rela

tive s

urf

ace c

overa

ge

CuDTTCT+FeDTTCT loading (mg)

Control (0.25+0.25) (0.5+0.5) (0.75+0.75)

*

Control

(0.25+0.25)

(0.5+0.5)

(0.75+0.75)

Chapter 5

133

Table 5.1 Corresponding ICP-OES measurements of leached copper and iron ions from

the mixed metal films after 3 days incubation in 2 mL of nitrifying bacteria culture.

Samples Leached copper

(mg/L)

Leached iron

(mg/L)

%mass of copper

leached out

%mass of iron

leached out

0.25+0.25 0.11 0.34 0.27 0.82

0.5+0.5 0.21 0.28 0.25 0.40

0.75+0.75 0.40 0.12 0.29 0.09

The effect of metals toxicity was also investigated by testing individual copper

or iron films alone (along with the corresponding leached soluble species). Overall, the

presence of up to 2 mg FeDTTCT (with 0.6 mg/L leached Fe, Table 5.2) alone, or up to

2 mg CuDTTCT (with 1.2 mg/L leached Cu, Table 5.3) alone did not alter the extent of

biofilm growth and the presence of planktonic biomass relative to the control (Figure

5.7). Therefore, this ‘dispersal-type’ antibiofilm activity does not originate from metal

toxicity, but most likely result from the presence of NO, which seems to be catalytically

generated by the mixed metal complexes from nitrite endogenously produced by the

AOB biofilm.

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134

Figure 5.7 Protein measurements of nitrifying bacteria biomass grown in the presence of

various loading of (a) FeDTTCT films and (b) CuDTTCT films. All values shown are

normalised to the bacteria biomass grown in the presence of bare film (control, inset).

Error bars indicate standard error between replicates (n = 4); *p ≤ 0.05 against the

control.

Table 5.2 Corresponding ICP-OES measurements of leached iron ions from the FeDTTCT

films after 3 days incubation in 2 mL of nitrifying bacteria culture.

FeDTTCT loading

(mg) Leached iron (mg/L)

%mass of iron

leached out

0.1 0.58 3.37

0.25 0.59 1.37

0.5 0.59 0.69

1 0.52 0.30

2 0.55 0.16

0.0

0.5

1.0

1.5

2.0

2.5

*

Rela

tive p

rote

in c

oncentr

ation

CuDTTCT loading (mg)

Biofilm

Planktonic

Control 0.1 0.25 0.5 1 2

0

100

200

Pro

tein

co

nc

en

tra

tio

n

(µg

/mL

)

Control

0.0

0.5

1.0

1.5

2.0

2.5

Rela

tive p

rote

in c

oncentr

ation

FeDTTCT loading (mg)

Biofilm

Planktonic

Control 0.1 0.25 0.5 1 2

0

100

200

Pro

tein

co

nc

en

tra

tio

n

(µg

/mL

)

Control

(a)

(b)

Chapter 5

135

Table 5.3 Corresponding ICP-OES measurements of leached copper ions from the

CuDTTCT films after 3 days incubation in 2 mL of nitrifying bacteria culture.

CuDTTCT loading

(mg) Leached copper (mg/L)

%mass of copper

leached out

0.1 0.08 0.46

0.25 0.45 1.03

0.5 0.71 0.81

1 0.87 0.50

2 1.18 0.34

To confirm the involvement of the catalytically generated NO in the observed

antibiofilm activity, the well-characterised NO scavenger PTIO, which has been

commonly used to provide insight into the physiological function of NO,20,21 was added

into the mixed metal systems. The scavenging activity of 100 µM PTIO towards

catalytically generated NO (from added nitrite) in the mixed metal

CuDTTCT+FeDTTCT systems was validated before (Figure 5.3). The PTIO

concentration chosen was found to have minimal effect on biofilm growth (Figure 5.8a),

which was also reported in earlier studies.20 Here, the presence of 100 µM PTIO in the

mixed metal systems were found to eliminate the antibiofilm effects previously seen

with the (0.5+0.5) mg or (0.75+0.75) mg mixed metal complexes, as comparable

amount of biofilm and planktonic biomass as those of the control growth was observed

(p-value = 0.99 vs the control or control+PTIO; Figure 5.8b).

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136

Figure 5.8 Protein measurements of nitrifying bacteria biomass grown in the presence of

(a) various PTIO concentration and (b) various loading of mixed metal films with the

addition of PTIO (100 µM). All values shown are normalised to the bacteria biomass

grown in the presence of bare film (control, inset). Error bars indicate standard error

between replicates (n = 4); *p ≤ 0.05 against the control.

Taken together, the findings show the capability of the CuDTTCT+FeDTTCT

mixed metal complexes to catalytically convert nitrite that was endogenously produced

by nitrifying bacteria biofilm, to NO. Subsequently, the NO generated suppressed the

biofilm formation, while confining bacterial growth to the planktonic phase. Such NO-

0.0

0.5

1.0

1.5

2.0

Re

lative

pro

tein

co

nce

ntr

atio

n

CuDTTCT+FeDTTCT loading (mg)

Biofilm

Planktonic

Control Control

+PTIO

(0.25+0.25)

+PTIO

(0.5+0.5)

+PTIO

(0.75+0.75)

+PTIO

0

100

200

300

Pro

tein

co

nc

en

tra

tio

n

(µg

/mL

)

Control

0.0

0.5

1.0

1.5

2.0

Rela

tive p

rote

in c

oncentr

ation

PTIO concentration (µM)

Biofilm

Planktonic

*

Control 50 100 200

0

100

200

Pro

tein

co

nc

en

tra

tio

n

(µg

/mL

)

Control

(a)

(b)

Chapter 5

137

mediated biofilm dispersal has been reported in a wide spectrum of biofilm-forming

microorganisms.22–25 A common mechanism underlying the transition from biofilm to

planktonic phase is via the NO-regulated alteration of intracellular levels of the

secondary messenger cyclic di-GMP (c-di-GMP), already observed in various bacteria,

including Pseudomonas aeruginosa and Shewanella oneidensis.26–28 In general, low

cellular c-di-GMP levels tend to induce biofilm dispersal, activate cell’s motility and

promote planktonic growth, while an increase in cellular c-di-GMP level promotes cell

attachment to surfaces and biofilm formation.29 In Nitrosomonas europaea, which is

one of the major members of AOB, the link between c-di-GMP signaling and NO-

mediated dispersal has yet been fully established. Instead, NO-mediated dispersal in the

bacteria has been shown to involve up-regulations of 11 proteins, such as motility

(flagellar) and flagellar assembly proteins.30 Due to the involvement of signalling

pathways in NO-mediated dispersal, cellular membrane damage is not expected to

occur, as confirmed by confocal images shown earlier (Figure 5.6c-f, no red (dead) cells

were observed). Such ‘non-toxic’ mode of biofilm dispersal, often associates with a

unique characteristic of NO release, as described in the following.

The antibiofilm effects were observed over relatively long period of 3 days and

given the short half-life of NO in biological systems, this suggests the continual slow

release of low concentrations of NO by the mixed CuDTTCT+FeDTTCT metal

systems. The slow release is a likely consequence of interactions between the redox

cycling of the complexes’ metallic centres and the activity of the slow growing AOB as

nitrite producer. Further, the iron centre could act as an electron mediator that receive

electrons from bacteria,1 such as Bacillus spp (also present in the bacterial inoculum),

which have been shown to utilise (“breathe”) insoluble substrates such as iron(III).31

Such cellular respiration can occur via secretion and subsequent transfer of extracellular

Chapter 5

138

redox metabolites from the bacteria known as electron shuttles, to the iron substrate, or

alternatively can involve direct contact between cell appendages (e.g. pili proteins or

EPS components) with the iron substrate.32 These effects could restart the redox cycling

of iron and copper and subsequently, the generation of NO from nitrite produced by the

AOB. As observed here, the indicated slow release of NO at low concentrations tend to

induce a dispersal effect converting bacteria to the free-floating planktonic phase, which

will otherwise induce lethal effects on biofilms if rapidly generated at high levels.33–35

Due to its non-growth inhibiting or non-cell killing antibiofilm effect, the development

of bacterial resistance to NO is unlikely.36,37 This importantly implies minimal

emergence of highly tolerant biofilm with the use of CuDTTCT+FeDTTCT metal

complexes as NO-based antibiofilm technology.

5.4. Summary

An iron complex (FeDTTCT) was successfully synthesised and incorporated

into PVC matrix together with an active copper species (CuDTTCT) for catalytic

generation of NO from nitrite. Amperometric measurement showed that the mixed

metal complexes (CuDTTCT+FeDTTCT) in nitrite solution was able to generate NO

without additional reducing agent. NO was generated from nitrite through

copper(II)/copper(I) redox cycling, whereby nitrite reduction to NO is via reaction with

copper(I), the latter was formed from reduction of copper(II) facilitated by the iron

complex, as shown by XPS analysis. It was found here that the mixed metal complexes

were capable of converting nitrite that was endogenously produced by nitrifying

bacteria biofilms, into NO. The generated NO subsequently exhibited a ‘dispersal-type’

antibiofilm activity or in other words, suppressing the biofilm formation without

Chapter 5

139

inhibiting planktonic bacterial growth. The involvement of NO in the antibiofilm

activity was validated by the use of NO scavenger, whereby its presence in the mixed

metal system reverted the antibiofilm effects.

5.5. References

1. Zhang, C. et al. Insoluble Fe-humic acid complex as a solid-phase electron

mediator for microbial reductive dechlorination. Environ. Sci. Technol. 48, 6318–

6325 (2014).

2. Welch, K. D., Davis, T. Z., van Eden, M. E. & Aust, S. D. Deleterious Iron-

Mediated Oxidation of Biomolecules. Free Radic. Biol. Med. 32, 577–583

(2002).

3. Liu, Z. D. & Hider, R. C. Design of iron chelators with therapeutic application.

Coord. Chem. Rev. 232, 151–171 (2002).

4. Hwang, S. Nitric Oxide Generating Polymers (NOGPs). (The University of

Michigan, 2007).

5. Breviglieri, S. T., Cavalheiro, E. T. G. & Chierice, G. O. Correlation between

ionic radius and thermal decomposition of Fe (II), Co (II), Ni (II), Cu (II) and Zn

(II) diethanoldithiocarbamates. Thermochim. Acta 356, 79–84 (2000).

6. Yang, Y., Ding, H., Hao, S., Zhang, Y. & Kan, Q. Iron(III), cobalt(II) and

copper(II) complexes bearing 8-quinolinol encapsulated in zeolite-Y for the

aerobic oxidation of styrene. Appl. Organomet. Chem. 25, 262–269 (2011).

7. Saghatforoush, L. A., Aminkhani, A. & Chalabian, F. Iron(III) Schiff base

complexes with asymmetric tetradentate ligands: synthesis, spectroscopy, and

antimicrobial properties. Transit. Met. Chem. 34, 899–904 (2009).

8. Lundberg, J. O., Weitzberg, E. & Gladwin, M. T. The nitrate-nitrite-nitric oxide

pathway in physiology and therapeutics. Nat. Rev. Drug Discov. 7, 156–167

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(2008).

9. Opländer, C. et al. Redox-mediated mechanisms and biological responses of

copper-catalyzed reduction of the nitrite ion in vitro. Nitric Oxide 35, 152–164

(2013).

10. McCarthy, C. W., Guillory II, R. J., Goldman, J. & Frost, M. C. Transition Metal

Mediated Release of Nitric Oxide (NO) from S-Nitroso-N-acetylpenicillamine

(SNAP): Potential Applications for Endogenous Release of NO on the Surface of

Stents via Corrosion Products. ACS Appl. Mater. Interfaces 8, 10128–10135

(2016).

11. Bartha, B., Kolbert, Z. & Erdei, L. Nitric oxide production induced by heavy

metals in Brassica juncea L. Czern and Pisum sativum L. Acta Biol. Szeged. 49,

9–12 (2005).

12. Oh, B. K. & Meyerhoff, M. E. Catalytic generation of nitric oxide from nitrite at

the interface of polymeric films doped with lipophilic Cu(II)-complex: a potential

route to the preparation of thromboresistant coatings. Biomaterials 25, 283–293

(2004).

13. Matocha, C. J., Karathanasis, A. D., Rakshit, S. & Wagner, K. M. Reduction of

copper(II) by iron(II). J. Environ. Qual. 34, 1539–1546 (2005).

14. Tao, L. & Li, F. Electrochemical evidence of Fe(II)/Cu(II) interaction on titanium

oxide for 2-nitrophenol reductive transformation. Appl. Clay Sci. 64, 84–89

(2012).

15. González, A. G., Pérez-Almeida, N., Magdalena Santana-Casiano, J., Millero, F.

J. & González-Dávila, M. Redox interactions of Fe and Cu in seawater. Mar.

Chem. 179, 12–22 (2016).

16. Frierdich, A. J. & Catalano, J. G. Fe(II)-mediated reduction and repartitioning of

structurally incorporated Cu, Co, and Mn in iron oxides. Environ. Sci. Technol.

46, 11070–11077 (2012).

17. O’Loughlin, E. J., Kelly, S. D., Kemner, K. M., Csencsits, R. & Cook, R. E.

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Reduction of AgI, AuIII, CuII, and Hg II by FeII/FeIII hydroxysulfate green rust.

Chemosphere 53, 437–446 (2003).

18. Grosvenor, A. P., Kobe, B. A., Biesinger, M. C. & McIntyre, N. S. Investigation

of multiplet splitting of Fe 2p XPS spectra and bonding in iron compounds. Surf.

Interface Anal. 36, 1564–1574 (2004).

19. Yamashita, T. & Hayes, P. Analysis of XPS spectra of Fe2+ and Fe3+ ions in

oxide materials. Appl. Surf. Sci. 254, 2441–2449 (2008).

20. Sauder, L. A., Ross, A. A. & Neufeld, J. D. Nitric oxide scavengers differentially

inhibit ammonia oxidation in ammonia-oxidizing archaea and bacteria. FEMS

Microbiol. Lett. 363, 1–7 (2016).

21. Goldstein, S., Russo, A. & Samuni, A. Reactions of PTIO and carboxy-PTIO

with *NO, *NO2, and O2-*. J. Biol. Chem. 278, 50949–50955 (2003).

22. Barraud, N. et al. Nitric oxide-mediated dispersal in single- and multi-species

biofilms of clinically and industrially relevant microorganisms. Microb.

Biotechnol. 2, 370–378 (2009).

23. Craven, M. et al. Nitric Oxide-Releasing Polyacrylonitrile Disperses Biofilms

Formed by Wound-Relevant Pathogenic Bacteria. J. Appl. Microbiol. 120, 1085–

1099 (2016).

24. Arora, D. P., Hossain, S., Xu, Y. & Boon, E. M. Nitric Oxide Regulation of

Bacterial Biofilms. Biochemistry 54, 3717–3728 (2015).

25. Barnes, R. J. et al. Nitric oxide treatment for the control of reverse osmosis

membrane biofouling. Appl. Environ. Microbiol. 81, 2515–2524 (2015).

26. Barraud, N. et al. Nitric oxide signaling in Pseudomonas aeruginosa biofilms

mediates phosphodiesterase activity, decreased cyclic di-GMP levels, and

enhanced dispersal. J. Bacteriol. 191, 7333–7342 (2009).

27. Ha, D.-G. & O’Toole, G. A. c-di-GMP and its Effects on Biofilm Formation and

Dispersion: a Pseudomonas aeruginosa Review. Microbiol. Spectr. 3, MB–

0003–2014 (2015).

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28. Plate, L. & Marletta, M. A. Nitric Oxide Modulates Bacterial Biofilm Formation

through a Multicomponent Cyclic-di-GMP Signaling Network. Mol. Cell 46,

449–460 (2012).

29. Liu, S. et al. Understanding, Monitoring and Controlling Biofilm Growth in

Drinking Water Distribution Systems. Environ. Sci. Technol. DOI:

10.1021/acs.est.6b00835 (2016).

30. Schmidt, I., Steenbakkers, P. J. M., op den Camp, H. J. M., Schmidt, K. & Jetten,

M. S. M. Physiologic and Proteomic Evidence for a Role of Nitric Oxide in

Biofilm Formation by Nitrosomonas europaea and Other Ammonia Oxidizers. J.

Bacteriol. 186, 2781–2788 (2004).

31. Nealson, K. H. & Saffarini, D. Iron and manganese in anaerobic respiration:

environmental significance, physiology, and regulation. Annu. Rev. Microbiol.

48, 311–343 (1994).

32. Nealson, K. H. & Finkel, S. E. Electron flow and biofilms. MRS Bull. 36, 380–

384 (2011).

33. Barraud, N., Kelso, M. J., Rice, S. A. & Kjelleberg, S. Nitric Oxide : A Key

Mediator of Biofilm Dispersal with Applications in Infectious Diseases. Curr.

Pharm. Des. 21, 31–42 (2015).

34. Barraud, N. et al. Involvement of nitric oxide in biofilm dispersal of

Pseudomonas aeruginosa. J. Bacteriol. 188, 7344–7353 (2006).

35. Deupree, S. M. & Schoenfisch, M. H. Morphological analysis of the

antimicrobial action of nitric oxide on gram-negative pathogens using atomic

force microscopy. Acta Biomater. 5, 1405–1415 (2009).

36. Privett, B. J., Broadnax, A. D., Bauman, S. J., Riccio, D. A. & Schoenfisch, M.

H. Examination of bacterial resistance to exogenous nitric oxide. Nitric Oxide 26,

169–173 (2012).

37. Njoroge, J. & Sperandio, V. Jamming bacterial communication: New approaches

for the treatment of infectious diseases. EMBO Mol. Med. 1, 201–210 (2009).

143

Chapter 6.

Conclusions and Recommendations

6.1. Conclusions

The research presented herein aims to assess and develop a catalytic system that

can generate nitric oxide (NO) for nitrifying bacteria biofilm control in industrial water

system. A copper complex, namely CuDTTCT, was synthesised and embedded within a

PVC matrix and tested as an NO-generating catalyst in the presence of ascorbic acid as

a reducing agent and nitrite as an NO source. The copper(II) complex was reduced to

copper(I) by ascorbic acid, which subsequently catalysed the reduction of nitrite to NO

while being oxidised back to copper(II). Enhanced and prolonged NO production was

observed when CuDTTCT was used compared to the addition of nitrite-ascorbic acid

alone. The system was found to be effective in suppressing the formation of nitrifying

bacteria biofilm, where it was revealed that the observed biofilm suppression is

predominantly caused by produced NO. Significant cell death was not observed,

indicating that prior to biofilm formation, the NO generated acts as a cell proliferation

inhibitor. Induction of NO production by the copper complex system also triggered the

dispersal of pre-formed biofilms. Additionally, simultaneous biofilm dispersal and

biofilm cells killing were observed when a high concentration of nitrite-ascorbic acid (5

mM) was used along with the CuDTTCT catalyst. Therefore, mechanism of action of

NO is complex and can occur via multiple pathways depending on the biofilm growth

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stage. This study highlights the potential of catalytically generated NO for nitrifying

bacteria biofilm control.

In the subsequent study, the ubiquitous Fe2+ ions were employed as an

alternative reducing agent for initiating the copper(II)/copper(I) redox cycling. The

combination of CuDTTCT-nitrite-Fe2+ was found to be capable of NO generation.

However, the amount of NO generated is highly dependent on the Fe2+ speciation in

different pH condition and importantly, buffer composition. The presence of

complexing agents such as phosphate, carbonate, and EDTA was found to be a more

prominent factor in influencing Fe2+ autoxidation compared to pH. Nonetheless, the

system is still effective in generating sufficient NO for biofilm dispersal and reduction

of cell aggregations. Comparing the system used in this study and that used in Chapter

3, these result thus imply that biofilm dispersal mediated by NO is not affected by the

selection of reducing agent. Importantly, buffer condition was also found to have

minimal impact on NO-mediated dispersal, thus it may have a higher possibility of

success when applied in real water condition. The findings of this study hence

emphasised the robustness of NO-mediated dispersal method for biofilm control.

In order to eliminate the need for the addition of a reducing agent in solution

form, an iron complex, namely FeDTTCT, was synthesised and incorporated into the

PVC matrix with the CuDTTCT catalyst. FeDTTCT was shown to facilitate

copper(II)/copper(I) redox cycling, thus expediting NO generation from nitrite without

the need for additional reducing agent. Moreover, the mixed metal complexes system

was capable of suppressing biofilm formation while confining bacteria growth to the

free-floating planktonic phase over three days incubation, which suggests the

conversion of nitrite endogenously produced by the nitrifying bacteria to NO.

Importantly, this ‘dispersal-type’ antibiofilm activity does not involve cell inhibition or

Chapter 6

145

cell killing pathway, and therefore could minimise the possible emergence of bacterial

resistance. The involvement of NO in exerting antibiofilm activity was confirmed by the

addition of an NO scavenger, whereby its presence with the mixed metal system

reverted the antibiofilm effect. This work highlights the development of self-sustained

metal-based material that could be potentially applied in industrial water systems.

6.2. Recommendations

As mentioned earlier, the presence of reducing agents is paramount in the

copper(II)/copper(I) redox cycling for NO generation. The studies presented here show

the use of ascorbic acid, Fe2+ ions, and iron complex as reducing agents. In addition to

the above, there are numerous attractive reducing agent candidates that can be tested.

For instance, Ren et al. reported the use of cathodic potentials as an alternative means to

reduce copper(II)-tri(2-pyridylmethyl)amine (Cu-TPMA) within silicone rubber

catheters to generate NO from a nitrite reservoir.1,2 Moreover, it was found that

copper(I) species can be generated from the reduction of copper(II) species by hydrogen

peroxide, which is produced from photochemical reactions of organic compounds in

natural waters.3,4 Other possible reducing agents include hydroxylamine5 and glucose.6

The study on effectiveness and suitability of different reducing agents is crucial to assist

in understanding the selection criteria for suitable reducing agents for

copper(II)/copper(I) redox cycling.

The study presented here also proved that NO can trigger biofilm dispersal on

the model organism nitrifying bacteria employed here. Although it was mentioned that

regulation of motility expression has been linked as the underlying mechanism involved

Chapter 6

146

for the switch between biofilm to planktonic phase in Nitrosomonas europaea,7 which is

one of the major member of ammonia oxidisers, the underlying mechanism for the test

bacteria used in this study, specifically Nitrosospira multiformis, Nitrospira marina, and

Bacillus sp. has yet to be clarified. Proteomic approach, which has been used by

Schmidt et al. to investigate protein regulation in different phase of bacteria cells,7 can

be used as the first step towards in-depth understanding of NO-mediated dispersal in the

test organisms used in study. Moreover, measurement of the intracellular level of cyclic-

di-GMP is suggested, as it is believed that this secondary messenger molecule is the

central mechanism involved in regulating biofilm dispersal in many bacterial species.8–

10

In this study, nitrifying bacteria are chosen because they are commonly found in

chloraminated water.11 While this study is aimed for the application in various industrial

water system, biofilm-forming bacteria can go beyond nitrifying bacteria group. For

instance, Legionella is commonly found in air conditioners, water systems, cooling

towers, water fountains, and hospital equipments.12 In membrane systems,

Sphingomonas,13 Acinetobacter,14 and Staphylococcus14 have been identified as the

pioneer biofilm-forming bacteria. Moreover, anaerobic bacteria such as Desulfovibrio

would be the dominant species in water systems where oxygen is not readily available.

Therefore, it is suggested that biofilm control by catalytically generated NO can be

tested against wider water-related bacterial consortiums. Additionally, antibiofilm

activity testing against natural occurring microbial consortiums formed from real water

sample would be of interest.

The study presented in Chapter 5 shows the use of a mixed metal-based material

for biofilm control for a period of three days. However, studies have reported that

increased biofilm resistance is observed in mature/thicker biofilms than early

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stage/thinner biofilms.15 Therefore, investigation over longer incubation periods (up to

one week) is recommended. In addition, continuous systems that will allow thicker

biofilm formation, such as flow cells, can be used to study the impact of biofilm

thickness (maturity) against the mixed metal system effectivity.

Although the mixed metal system has been proven to be effective in suppressing

nitrifying biofilm formation in a non-toxic manner, the underlying mechanism, i.e. the

NO release profile and NO effective concentration is yet to be established. The widely

employed chemiluminescence for prolonged measurement of NO can be used to

understand NO release profile from the mixed metal system during the incubation

period.16 The effective concentration of NO that affected the cells can be measured by

employing the intracellular NO-sensitive dye, 4,5-diaminofluorescein diacetate (DAF-2

DA).17,18 Additionally, in-depth understanding on the postulated mechanism for

prolonged NO mechanism presented in Chapter 5, whereby the oxidised iron complex

was reduced via extracellular microbial respiration, can be investigated. The above

knowledge will allow better understanding towards the applicability of the mixed metal

catalyst system for long-term application.

Barraud et al. reported that biofilms which receive treatment with NO donors

exhibit higher sensitivity towards chlorine.19 Thus, investigation on the combined

biofilm removal efficiency of catalytically generated NO with chlorine or chloramine is

also suggested. It should be noted that chlorine/chloramine, which are oxidants, can

neutralise any reducing agents available in water. Therefore, the study on the mixed

metal system developed in Chapter 5 can be extended with this investigation.

For future applications, it is important to study industrial scale production of

NO-generating materials which does not involve the use of organic solvent as shown in

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this thesis. Consequently, the production of PVC-copper complex material by the

commonly used extrusion method is recommended. Because a simple physical mixing

process between the PVC and the metal complexes will be sufficient for the

incorporation of the metal complexes, the cost associated with the synthesis of PVC

containing metal complex is expected to be minimal. Nevertheless, economic

assessment regarding this process is needed.

Future research also should be directed towards the design of novel ligands that

can increase the stability of the metal centre to prevent metal ion leaching (as mentioned

in Chapter 3) and prevent aggregation of the metal complexes (especially iron complex)

in PVC matrix. Moreover, novel ligand design that can ‘mask’ copper species de-

stabilising influence towards the polymeric chain without affecting the

copper(II)/copper(I) redox activity. This research could also be expanded with studies

on other transition metals that can catalytically reduce nitrite to NO and have higher

compatibility with PVC. For instance, cobalt, nickel, or zinc have been found to mediate

NO generation from S-nitroso-N-acetylpenicillamine (SNAP; a synthetic NO donor).20

In the long term, the materials developed in this study can be easily adapted to

different applications with minor adjustments. For instance, in clinical settings, the use

of glutathione (a reducing agent commonly available in plasma and blood) to reduce the

copper complex used in this study can be investigated. It is also recommended to test

NO-generating materials effectivity against some targeted pathogens such as

Pseudomonas aeruginosa, Staphylococcus aureus, and Escherichia coli.

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