optimization of cultivation conditions for e. coli biofilm formation … · 2017-08-28 · as a...

Optimization of cultivation conditions

for E. coli biofilm formation

in microtiter plates

Luciana Calheiros Gomes

Dissertation for Master's degree in Bioengineering

Supervised by Professor Filipe Mergulhão

Department of Chemical Engineering

Faculty of Engineering, Porto University

July 2011

When you make the finding yourself — even if you’re the

last person on Earth to see the light — you’ll never forget it.

(Carl Sagan)

Optimization of cultivation conditions for E. coli biofilm formation in microtiter plates

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ACKNOWLEDGMENTS

Quero aqui expressar os meus agradecimentos a todos os que me apoiaram,

ajudaram e encorajaram ao longo deste projecto.

Ao LEPAE (Laboratório de Engenharia de Processos, Ambiente e Energia) por ter

facultado os recursos necessários para a realização desta dissertação.

Ao meu orientador, Professor Filipe Mergulhão, pela enorme disponibilidade e

atenção dedicada a este mestrado.

Ao Professor Manuel Simões, por ter partilhado comigo a sua experiência com os

métodos de quantificação de biofilme.

Aos meus colegas do Mestrado Integrado em Bioengenharia, especialmente à Rita e

à Ana pela sua amizade e ajuda.

Aos colegas do laboratório E303 e E204, pelo óptimo convívio e, em particular,

gostaria de agradecer à Joana M., por ter dividido comigo a tarefa "infindável" das

microplacas, e à Joana T. pelo encorajamento.

Aos meus Pais e Irmão por estarem sempre presentes.

Ao J. P. pelo amor, preocupação e carinho ao longo destes meses.


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ABSTRACT

The attachment of bacterial cells to solid surfaces and its subsequent growth and production of

extracellular polymeric substances (EPS) forms a biofilm. Its establishment and development are dynamic and

complex processes that are regulated by diverse characteristics of the growth medium, substratum and cell

surface. Among diverse parameters, nutrient composition and hydrodynamic conditions were the

environmental factors studied in this thesis. The goal was to evaluate the effect of nutrient concentration in

biofilm formation under different agitation conditions. Escherichia coli, a Gram-negative bacterium, was used

as a model organism and a 96-well microtiter plate was the platform chosen for this biofilm study.

The cultivation conditions included two orbital shaking diameters (25 and 50 mm) at the same

frequency (150 rpm) and no shaking (0 rpm). The effect of three concentrations of the following nutrients was

studied: glucose (0.25, 0.5 and 1 g.L-1), peptone (0.25, 0.5 and 1 g.L-1) and yeast extract (0.125, 0.5 and 1 g.L-

1). Based on the results obtained in the study of the individual variation of each nutrient, we tried to find a

culture media composition that would generate more biofilms in dynamic conditions. Eight combinations of the

three components were tested in both orbital diameters under a shaking frequency of 150 rpm.

The results obtained upon glucose variation indicate that the amount of E. coli biofilm produced

increased with increasing glucose concentrations for tested cultivation conditions. In dynamic conditions, the

maximum biofilm value was reached at 24 hours for the most concentrated medium (1 g.L-1). The highest

glucose consumption occurred during the first 24 hours for all media. However, this nutrient has never been

depleted after 60 hours for the highest glucose concentration.

Looking at the peptone results, biofilm formation was higher in the highest peptone concentration

conditions (1 g.L-1) when an orbital shaker with a diameter of 50 mm at 150 rpm was used. There was no

difference in the amount of attached cells when varying the yeast extract concentration in the culture medium

in the range of concentrations and cultivation conditions used.

In opposition to the crystal violet assay which correlates to the amount of biofilm formed, the results of

resazurin assay which are indicative of the metabolic state of the cells were inconclusive.

Optimization tests seem to indicate that glucose is the parameter with greater influence on the

absorbance values obtained in the crystal violet method for both dynamic conditions under investigation. This

can be attributed to the higher amount of E. coli biofilms formed in microtiter plates or to the establishment of

more cohesive biofilms. A single optimized medium composition could not be found but three formulations

seem to increase the absorbance signals: a) 1 g.L-1 glucose, 0.5 g.L-1 peptone and 1 g.L-1 YE; b) 1 g.L-1 glucose,

1 g.L-1 peptone and 0.5 g.L-1 YE; c) 0.5 g.L-1 glucose, 0.5 g.L-1 peptone and 1 g.L-1 YE. In these experiments,

the main carbon source was exhausted after 36 hours, probably due to bacterial growth.

The shaken 96-well microtiter plate is a valuable platform for the screening of different parameters

involved in E. coli biofilm formation.

Keywords: Escherichia coli; biofilm; nutrient concentration; orbital shaking; microtiter plate; crystal

violet; resazurin.


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RESUMO

A adesão de células bacterianas a superfícies sólidas e o seu consequente crescimento e produção de

substâncias poliméricas extracelulares (EPS) cria um biofilme. A origem e desenvolvimento de um biofilme são

processos dinâmicos e complexos regulados por diversas características do meio de cultura, material onde adere e

superfície celular. De entre os diversos parâmetros, a composição nutricional e as condições hidrodinâmicas foram os

factores ambientais estudados nesta tese. O principal objectivo foi avaliar o efeito da concentração de nutrientes na

formação de biofilmes sujeitos a diferentes condições de agitação. Utilizou-se como organismo modelo a bactéria

Gram-negativa Escherichia coli e como plataforma de estudo dos biofilmes a microplaca de 96 poços.

Quanto às condições de incubação, foram testados dois diâmetros de agitação orbital (25 e 50 mm) à mesma

frequência (150 rpm) e um deles parado (0 rpm). Avaliou-se o efeito de três concentrações dos seguintes nutrientes

do meio de cultura: glucose (0.25, 0.5 e 1 g.L-1), peptona (0.25, 0.5 e 1 g.L-1) e extracto de levedura (0.125, 0.5 e 1

g.L-1). Com base nos resultados obtidos neste estudo da variação individual de cada nutriente, tentou-se chegar a uma

formulação do meio de cultura que originasse maior quantidade de biofilme em condições dinâmicas. Testou-se oito

combinações dos três componentes do meio em ambos os diâmetros de agitação a 150 rpm.

Os resultados obtidos aquando da variação da glucose indicam que quanto maior for a concentração de

glucose, maior é a quantidade de biofilme que se forma para as condições de incubação testadas. Em condições

dinâmicas, a quantidade máxima de biofilme foi atingida às 24 horas para o meio mais concentrado (1 g.L-1). O maior

consumo de glucose deu-se durante as primeiras 24 horas da experiência em todas as concentrações, sendo que, para

a concentração mais elevada de glucose, este nutriente nunca foi totalmente consumido após 60 horas.

Relativamente aos ensaios em que se variou a concentração de peptona, a formação de biofilme foi maior no

meio mais rico em peptona (1 g.L-1), para o agitador de maior amplitude. Nos ensaios que se testou o efeito do

extracto de levedura, concluiu-se que a variação da concentração deste nutriente do meio não tem grande impacto, na

gama de concentrações testada e nas condições de agitação utilizadas.

Contrariamente ao método do violeta de cristal, que indica a quantidade de biofilme formado, os resultados

da resazurina (que são indicativos da actividade metabólica do biofilme) não foram conclusivos.

Os ensaios de optimização efectuados parecem indicar que a glucose é o parâmetro com maior influência nos

valores de absorvância obtidos no método do violeta de cristal em ambas as condições dinâmicas experimentadas.

Isto pode dever-se a maior quantidade de biofilmes de E. coli formados em microplacas ou ao estabelecimento de

biofilme mais coeso. Não foi possível determinar a composição óptima do meio que conduzisse à maior produção de

biofilme à escala laboratorial, mas três das formulações parecem aumentar os sinais de absorvância: a) 1 g.L-1

glucose, 0.5 g.L-1 peptona e 1 g.L-1 extracto de levedura; b) 1 g.L-1 glucose, 1 g.L-1 peptona e 0.5 g.L-1 extracto de

levedura; c) 0.5 g.L-1 glucose, 0.5 g.L-1 peptona e 1 g.L-1 extracto de levedura. Nestas experiências, a principal fonte

de carbono esgotou-se cerca de 36 horas depois, provavelmente porque foi utilizada no crescimento bacteriano.

A microplaca de 96 poços sob agitação é uma plataforma útil para a triagem de diversos parâmetros

envolvidos na formação de biofilmes de E. coli.

Palavras-chave: Escherichia coli; biofilme; concentração de nutrientes; agitação orbital; microplaca; violeta

de cristal; resazurina.


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LIST OF CONTENTS

ACKNOWLEDGMENTS ................................................................................................. i

ABSTRACT ..................................................................................................................... ii

RESUMO ........................................................................................................................ iii

LIST OF CONTENTS ..................................................................................................... iv

LIST OF FIGURES ......................................................................................................... vi

LIST OF TABLES .......................................................................................................... ix

LIST OF SYMBOLS ........................................................................................................ x

1 INTRODUCTION ..................................................................................................... 1

1.1 Objectives of experimental work .................................................................................. 1

1.2 Relevance of the work ................................................................................................... 1

1.3 Thesis outline ................................................................................................................ 2

2 LITERATURE REVIEW .......................................................................................... 5

2.1 Microbial biofilms ......................................................................................................... 5

2.2 The impact of biofilm formation ................................................................................... 7

2.3 Biofilm formation process ............................................................................................. 7

2.4 Parameters involved in the biofilm life cycle .............................................................. 10

2.4.1 Hydrodynamics ................................................................................................... 11

2.4.2 Nutrient availability ............................................................................................. 11

2.4.3 Hydrodynamic versus nutrient effects ................................................................. 13

2.5 Platforms for in vitro biofilm studies .......................................................................... 14

2.5.1 Microtiter plates .................................................................................................. 14

2.5.1.1 Recent applications ................................................................................ 17

2.5.1.2 Effect of orbital shaking on biofilm formation ...................................... 20

2.5.1.3 Biofilm quantification ............................................................................ 21

2.5.2 Other common platforms .................................................................................... 23

3 MATERIALS AND METHODS ............................................................................ 27

3.1 Microorganism ............................................................................................................ 27

3.2 Growing the cellular culture and preparation of inocula ............................................. 27

3.3 Biofilm formation system ............................................................................................ 28

3.3.1 Effect of individual variation of glucose concentration ...................................... 28

3.3.2 Effect of individual variation of the peptone concentration ................................ 30

3.3.3 Effect of individual variation of the YE concentration ....................................... 30


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3.3.4 Effect of culture medium formulations ............................................................... 31

3.4 Biofilm monitoring and quantification ........................................................................ 32

3.4.1 Crystal violet assay (CV assay) ........................................................................... 33

3.4.2 Resazurin assay ................................................................................................... 33

3.5 Glucose quantification ................................................................................................. 34

3.6 Statistical analysis ....................................................................................................... 35

4 RESULTS AND DISCUSSION ............................................................................. 37

4.1 Effect of individual variation of glucose, peptone and YE concentrations ................. 37

4.2 Effect of culture medium formulations ....................................................................... 49

5 CONCLUSIONS AND PERSPECTIVES FOR FURTHER RESEARCH ............ 59

REFERENCES ............................................................................................................... 63

ANNEXES ..................................................................................................................... A1

ANNEX A: Methods for generating mixing effects in microtiter plates ............................... A2

ANNEX C: Commonly used flow displacement systems for biofilm studies ....................... A4

ANNEX D: Specifications of Peptone from Meat (peptic), granulated (Merck Microbiology Manual) .................................................................................................................................. A6

ANNEX E: Specifications of Yeast Extract, granulated (Merck Microbiology Manual) ...... A7

ANNEX F: Calibration curve of glucose ............................................................................... A8

ANNEX G: Calculation of the volumetric oxygen transfer coefficient ...................... A9

ANNEX H: Extra graphics with results of culture media formulations ............................... A10


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LIST OF FIGURES

Figure 1. Escherichia coli JM109 (SEM scanning picture) (Chiang et al., 2009). ....................... 6

Figure 2. Biofilm accumulation through time (based on (Melo and Flemming, 2010)). .............. 8

Figure 3. Schematic representation of biofilm development: 1 – Initial reversible attachment, 2 –

Irreversible attachment, 3 – Development of biofilm architecture, 4 – Maturation, 5 –

Dispersion of cells from the biofilm into the surrounding environment (adapted from (Monroe,

2007)). ........................................................................................................................................... 8

Figure 4. Two-dimensional nutrient concentration-flow velocity habitat domain diagram based

on observational and hypothetical considerations of mass transfer and shear on biofilm

morphotypes (Stoodley et al., 1998). .......................................................................................... 14

Figure 5. Illustrative photograph of polystyrene microtiter plates used on biofilm formation: a)

6-well microplate, b) 48-well microplate and c) 96-well microplate. ......................................... 16

Figure 6. Shaking pattern during orbital shaking at 300 rpm in a round microwell of 6.5 mm of a

96-low-well MTP: a) at a shaking amplitude of 25 mm (OTR=16 mmol L-1 h-1; reasonable

degree of mixing, non-turbulent) and b) at a shaking amplitude of 50 mm (OTR=32 mmol L-1 h-

1; good mixing up to the very bottom, non-turbulent) (Enzyscreen). .......................................... 21

Figure 7. Illustrative photograph of CV assay applied to a 96-well microplate. ......................... 22

Figure 8. Conversion of resazurin to resorufin by viable cells. The fluorescence produced is

proportional to the respiratory activity of cells (adapted from (Promega, 2011)). ...................... 23

Figure 9. Microplate layout for testing the effect of glucose concentration. .............................. 30

Figure 10. Microplate layout for optimization tests. ................................................................... 32

Figure 11. Experimental results for E. coli biofilm formed in three cultivation conditions (1 –

d0=50 mm, 150 rpm; 2 – d0=25 mm, 150 rpm; 3 – no shaking) with different glucose

concentrations ( - 0.25 g.L-1, - 0.5 g.L-1 and - 1 g.L-1). A – Crystal violet assay

(absorbance at 570 nm); B – Resazurin assay (fluorescence at λex: 570 nm and λem: 590 nm).

Error bars indicate standard deviations of three experiments. .................................................... 40


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d0=50 mm, 150 rpm; 2 – d0=25 mm, 150 rpm; 3 – no shaking) with different glucose

concentrations ( - 0.25 g.L-1, - 0.5 g.L-1 and - 1 g.L-1). C – Glucose concentration

(g.L-1). Error bars indicate standard deviations of three experiments. ........................................ 41


d0=50 mm, 150 rpm; 2 – d0=25 mm, 150 rpm; 3 – no shaking) with different peptone



Error bars indicate standard deviations of four experiments. ...................................................... 45


d0=50 mm, 150 rpm; 2 – d0=25 mm, 150 rpm; 3 – no shaking) with different yeast extract



Error bars indicate standard deviations of four experiments. ...................................................... 47

Figure 15. Photograph illustrative of the "white mass" formed at the bottom of the microplate

wells (d0=50 mm; 48 hours). Thick red arrows point the mass attached/deposited in some wells.

In medium without cells (C) and water, it was not observed. ..................................................... 51

Figure 16. Crystal violet results for E. coli biofilm amount in different media formulations (see

the detailed composition on Table 4) at 50 mm orbital shaking diameter and 150 rpm. The

media plotted on the left have 1 g.L-1 glucose, while those represented on the right side contain

0.5 g.L-1 glucose. Error bars indicate standard deviations of three experiments. ........................ 52


the detailed composition on Table 4) at 25 mm orbital shaking diameter and 150 rpm. The

media plotted on the left have 1 g.L-1 glucose, while those represented on the right side contain

0.5 g.L-1 glucose. Error bars indicate standard deviations of three experiments. ........................ 52

Figure 18. Effect of the combination of glucose and peptone in alternating concentrations:

media 3 and 4 include 1 g.L-1 glucose and 0.5 g.L-1 peptone, while media 2 and 6 have 0.5 g.L-1

glucose and 1 g.L-1 peptone (see the detailed composition on Table 4). Individual test includes

data from experiments of individual peptone variation (Figure 13-2A); the medium contains

0.55 g.L-1 glucose, 1 g.L-1 peptone and 0.125 g.L-1 YE. Error bars indicate standard deviations

of three experiments, except for the individual test that correspond to standard deviations of four

experiments. ................................................................................................................................ 54


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Figure 19. The culture media where were detected the highest and lowest absorbance values

under dynamic conditions (see the detailed compositions on Table 4). Error bars indicate

standard deviations of three experiments. ................................................................................... 55

Figure 20. Glucose concentration in different media formulations under dynamic conditions (see

the detailed compositions on Table 4). Error bars indicate standard deviations of three

experiments. ................................................................................................................................ 56

Figure 21. Microplate mixing tools (BioShake.com, 2010). ......................................................... 2

Figure 22. Surface of Calgary Biofilm Device pegs with biofilm (Wei et al., 2006). .................. 3

Figure 23. BioFlux high-throughput system for screening of flow biofilm viability and other

parameters: a) photograph of BioFlux system and b) schematic diagram showing the system

operation (adapted from (Benoit et al., 2010)). ............................................................................. 3

Figure 24. Schematic representation of a rotating annular reactor (Gjaltema et al., 1994). .......... 4

Figure 25. A large-scale flow cell reactor: a) illustrative photograph and b) schematic

representation of the experimental apparatus system (adapted from (Teodósio et al., 2011a)). ... 4

Figure 26. Illustrative photograph of the experimental apparatus system used to perform biofilm

formation on a small-scale flow cell reactor. ................................................................................ 5

Figure 27. Glucose concentration standard curve (linear regression: y=0.4382x; R2=0.9949). .... 8


the detailed compositions on Table 4) under dynamic conditions. The media plotted on the left

have 1 g.L-1 peptone, while those represented on the right side have 0.5 g.L-1 peptone. Error bars

indicate standard deviations of three experiments. ..................................................................... 10


the detailed compositions on Table 4) under dynamic conditions. The media plotted on the left

have 1 g.L-1 yeast extract, while those represented on the right side have 0.5 g.L-1 yeast extract.

Error bars indicate standard deviations of three experiments. .................................................... 11

Figure 30. Resazurin results for E. coli biofilm formed under dynamic conditions in different

media formulations (see the detailed compositions on Table 4). Error bars indicate standard

deviations of three experiments................................................................................................... 12


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LIST OF TABLES

Table 1. Important variables in cell attachment and biofilm formation (based on (Donlan, 2002))

..................................................................................................................................................... 10

Table 2. Examples of recent applications of microtiter plates in the biofilms field .................... 17

Table 3. Microtiter plate-based assays (adapted from (Azevedo et al., 2009)) ........................... 22

Table 4. Media compositions for optimization tests ................................................................... 31

Table 5. Effect of the shaking amplitude on mass transfer in microtiter plates .......................... 43

Table 6. Maximum absorbance values of the CV method for individual variation of glucose,

peptone and yeast extract, and respective concentration and time point ..................................... 48


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LIST OF SYMBOLS

Symbology

Initial specific surface area (m-1)

Final specific surface area (m-1)

Area (m2)

Bond number (dimensionless)

Shaking amplitude or diameter (m)

Microwell vessel diameter (m)

Diffusion coefficient (m2.s-1)

Froude number (dimensionless)

Gravitational constant (m.s-2)

Volumetric overall mass transfer coefficient (s-1)

Shaking frequency (s-1)

Reynolds number (dimensionless)

Schmidt number (dimensionless)

Sherwood number (dimensionless)

Volume (m3)

Wetting tension (mN.m-1)

Abbreviations

cAMP Cyclic Adenosine Monophosphate

CFD Computational Fluid Dynamics

CRP cAMP receptor protein

DAPI 4',6'-diamidino-2-phenylindole

DNS Dinitrosalicylic Acid

ELISA Enzyme Linked Immunosorbent Assay

EPS Extracellular Polymeric Substances

OD Optical Density

OTR Oxygen Transfer Rate

MTP Microtiter Plate

PCA Plate Count Agar


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PS Polystyrene

PVC Polyvinylchloride

SD Standard Deviation

SEM Scanning Electron Microscope

YE Yeast Extract

Greek Letters

Density (kg.m-3)

Viscosity (kg.m-1.s-1)


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1 INTRODUCTION

1.1 Objectives of experimental work

In the last decades, several studies have addressed important factors influencing

the formation and properties of biofilms. These included the characteristics of the

microbial species and strains, surface composition and roughness, medium composition,

and hydrodynamic features of the fluid (such as velocity and turbulence).

This work stems from a PhD project ongoing at the Department of Chemical

Engineering, Faculty of Engineering of University of Porto, where the effect of the

nutrient load on biofilm formation by E. coli was assessed by combining different

substrate feed concentrations and dilution rates in a flow cell reactor (Teodósio et al.,

2010).

The main goal of the investigation behind this thesis was to study the effect of

nutrient concentration in biofilm formation under different agitation conditions.

Escherichia coli, a Gram-negative bacterium, was used as a model organism. The use of

this bacterium is related to the fact that it typically forms undesirable biofilms in food-

processing environments and on water-distribution systems. The platform chosen for

biofilm study was the 96-well microtiter plate, since it allows the rapid development of

biofilm samples and the simultaneous test of diverse culture media, unlike the system

used by Teodósio (2010). The incubation conditions included two orbital shaking

diameters (25 and 50 mm) at the same frequency (150 rpm) and no shaking. The effect

of three concentrations of the following nutrients was studied: glucose (0.25, 0.5 and 1

g.L-1), peptone (0.25, 0.5 and 1 g.L-1) and yeast extract (YE) (0.125, 0.5 and 1 g.L-1).

Based on the results obtained in the study of the individual variation of each nutrient,

we tried to find the optimal medium composition for high biofilm production in

dynamic conditions through the combination of the three tested compounds.

1.2 Relevance of the work

An understanding on how E. coli can establish and survive in processing

environments and on water-distribution systems is essential to finding ways to prevent


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contamination. This study tries to provide clues regarding the conditions under which

this organism can attach and form biofilms, and in characterizing the process.

Effects of nutrients and shear stresses on biofilm formation by E. coli were

examined, since the nutrient content of the growth medium and the different

hydrodynamic conditions caused by orbital incubator rotation have been found to

regulate the development of biofilms in many organisms (Dewanti and Wong, 1995;

Bühler et al., 1998; Duetz and Witholt, 2001). However, this study represents the first

report on the effect of various nutrients as well as the orbital shaking diameter on the

ability of this microorganism to form biofilms in the wells of the microtiter plates.

Although there is some information about the effect of glucose levels on biofilm

production, little is known about the effect of varying nitrogen concentrations,

specifically peptone and yeast extract, in the same process. This type of research is also

innovative in introducing the effect of the agitation diameter on biofilm growth in

microplates. Usually, when microtiter plates are used as a simulation system,

researchers only report the shaking frequency, unknowing that the orbital shaking

diameter can sometimes have greater impact on hydrodynamic conditions than the

shaking frequency itself.

Beyond the applicability of this thesis in the real-life, optimization of these

cultivation parameters in a high-throughput format is important for future work in other

biofilm-forming platforms at laboratory scale, for instance flow cells.

1.3 Thesis outline

This thesis is divided in five sections. Section 1 shows the context, main

objectives and motivations for the development of this work. Section 2 encloses the

literature review, where the process of biofilm formation is described in more detail,

highlighting the role of hydrodynamics and medium composition as variables involved

in the biofilm life cycle. The literature review gives special attention to the system used

in this project to study the dynamic of biofilm formation – the microtiter plate –

including recent applications and the explanation of the effect of shaking parameters on

biofilm growth. In Section 3, the materials and methodologies used to perform all the

experimental work are fully described. The results are presented, correlated and


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discussed in Section 4. This section evaluates the effect of varying glucose, peptone and

yeast extract concentrations on E. coli biofilm formation. At the same time, it discusses

the influence of the orbital shaking amplitude on cell attachment to polystyrene flat-

bottomed microtiter plates. Towards the end, a discussion on the results relating to the

optimization of the culture medium in dynamic conditions is presented. Section 5 gives

an overview of the experimental work and presents some ideas and suggestions for

future work.


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2 LITERATURE REVIEW

2.1 Microbial biofilms

Biofilms can be described as aggregates of cells irreversibly attached to a surface

and embedded within a self-produced matrix of extracellular polymeric substances

(EPS) (Melo, 2003; Van Houdt and Michiels, 2005). A current new definition for a

biofilm also takes into consideration other physiological attributes of these sessile

organisms, including an altered growth rate and the fact that biofilm organisms

transcribe genes that planktonic organisms (single-cells that

are suspended and dispersed in an aqueous medium) of the same species do not

(Shunmugaperumal, 2010). It is estimated that more than 90% of microorganisms live

in a structured community of cells since they constitute a more efficient way of

surviving in hostile environments (Costerton, 1985; Simões et al., 2010b). Practically

there is no surface that cannot be colonized (Characklis and Marshall, 1990), from inert

materials to living tissue or cells.

The biofilm composition is dependent on environmental factors, such as

temperature, pH, pressure, nutrient composition and dissolved oxygen (Flemming,

1991; O'Toole et al., 2000). Although biofilm composition is not necessarily uniform,

the extracellular matrix is basically composed by water, a collection of microorganisms,

predominantly bacteria, and their excretion products (EPS) (Marshall, 1984; Allison,

2003). Biofilm is considered a very absorbent and porous structure because it is mostly

made of water (often with 90-99%) and has water channels and pores. The microbial

cells represent about 2-5% of biofilm matrix and extracellular polymeric substances

correspond to 1-2% of matrix composition (Sutherland, 2001).

EPS are composed of polysaccharides, proteins, phospholipids and nucleic acids,

as well as other polymeric substances hydrated to 85% to 95%. The extracellular

substances are responsible for the cohesion (keeping the cells attached to one other) and

the adhesion (to surfaces). The EPS matrix plays an important role in biofilm

maintenance, since it delays or prevents biocides and other antimicrobial agents from

reaching microorganisms within the biofilm, and modulates the nutrient concentration


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necessary for their survival (Pereira and Vieira, 2001; Sutherland, 2001; Allison, 2003;

Chmielewski and Frank, 2003).

From the microorganisms found in a biofilm, bacteria are the predominant group.

The high reproduction rates, great adaptability and production of extracellular

substances and structures are the main characteristics which give bacteria large capacity

for biofilm production (Characklis and Marshall, 1990). Pseudomonas, Bacillus,

Alcaligens, Flavobacterium and Staphylococcus are the most common biofilm

producers (Mattila-Sandholm and Wirtanen, 1992). However, biofilm formation has

been best studied in members of the Pseudomonas genus because they are amongst the

most diversified bacterial species in the environment, they are involved in medical

conditions like cystic fibrosis and they are known to be good biofilm producers (Simões

et al., 2008). Comparatively to the biofilm studies and characterization using

Pseudomonas species, little is known about the capacity of Escherichia coli to develop

biofilms. A member of the family Enterobacteriaceae, E. coli is a Gram-negative,

facultative anaerobic and non-sporulating bacterium. Cells are typically rod-shaped and

are about 2 µm long and 0.5 µm in diameter (Figure 1). E. coli is a natural component

of the gastro-intestinal flora and coexists with the human host, usually with mutual

benefit. Being typically of fecal origin, it is a good indicator of the sanitary quality of

water and of the food-processing environments (Van Houdt and Michiels, 2005;

Teodósio et al., 2010).

Figure 1. Escherichia coli JM109 (SEM scanning picture) (Chiang et al., 2009).


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2.2 The impact of biofilm formation

Bacterial biofilms can have advantageous or negative effects, depending on where

they build up (Melo, 2003). Biofilms find application in processes as diverse as

bioremediation (Singh et al., 2006), production of industrial chemicals (Qureshi et al.,

2005), wastewater treatment (Lazarova and Manem, 1995), removal of volatile

compounds from waste streams (Vinage and Rohr, 2003) or even generation of

electricity in microbial fuel cells (Clauwaert et al., 2007). Nevertheless, biofilms are

often unwanted and cause serious problems in industrial, environmental, food and

biomedical fields. Besides causing problems in cleaning and disinfection, biofilms may

cause energy losses and blockages in membrane systems, cooling water tubes and heat

exchange channels, phenomenon known as biofouling. The fouling problems lead,

ultimately, to an increase in the plant operating costs, as well as to public health

concerns and environmental impacts (Pereira et al., 2008; Melo and Flemming, 2010).

Biofouling is especially problematic in food industry, where the growth of

microorganisms and the deposit of by-products from their metabolism in food

equipment and pipelines can lead to economic losses due to food spoilage and

equipment corrosion. Additionally, biofilms formed on food contact surfaces can affect

the quality and safety of the foods because of the release of foodborne pathogens

(Kumar and Anand, 1998; Chmielewski and Frank, 2003; Simões et al., 2010b). The

biofilms enjoying the worst reputation are certainly those found in the health sector

since they are responsible for more than 60% of all microbial infections in humans

(Shunmugaperumal, 2010). Microorganisms can attach on lungs, teeth, implants and

urinary catheters (Costerton, 1985; Donlan, 2002).

2.3 Biofilm formation process

According to Bott (1993), the accumulation of biofilm is a natural process, which

follows a sigmoid pattern (Figure 2) as a result of a balance between a variety of

physical, chemical and biological processes that occur simultaneously.


- 8 -

Figure 2. Biofilm accumulation through time (based on (Melo and Flemming, 2010)).

A common model for the formation of a differentiated and mature bacterial

biofilm has been proposed and includes five different stages: (i) an initial reversible

attachment to a pre-conditioned surface, (ii) transition from reversible to irreversible

attachment, (iii) development of biofilm architecture, (iv) development of microcolonies

into a mature biofilm, and (v) dispersion of cells from the biofilm into the surrounding

environment (Kumar and Anand, 1998; Stoodley et al., 2002; Van Houdt and Michiels,

2005). Biofilm formation steps can be observed in Figure 3.

Figure 3. Schematic representation of biofilm development: 1 – Initial reversible attachment, 2 –

Irreversible attachment, 3 – Development of biofilm architecture, 4 – Maturation, 5 – Dispersion of cells

from the biofilm into the surrounding environment (adapted from (Monroe, 2007)).


- 9 -

Although a solid knowledge has been accumulated about the later stages of

biofilm formation, the initial stages of development are still poorly understood. It is

known that the initial conditioning biofilm is a very thin monolayer formed on the

adhesion surface, which will eventually be the docking place for the first reversibly

attached cells. The rate at which the conditioning film forms depends on the

concentration of organic molecules in the culture medium that contact with the

surface, the affinity of those molecules to the support and the hydrodynamic features

of the fluid, such as velocity and turbulence (Chamberlain, 1992). As previously stated,

the physical properties of the surface are also of capital importance for the adhesion of

organic molecules, namely the surface charge, free energy and roughness (Tsibouklis et

al., 1999; Boulange-Petermann et al., 2004; Carnazza et al., 2005).

After the initial conditioning film is established, there is the transport of microbial

cells from the aqueous medium to the solid surface. The molecules present in the initial

development of biofilm may provide a strong and stable adhesion through the formation

of polymeric chains with the exopolymers on the surface of microorganisms, or as a

consequence of motility that some cells present due to the existence of external

filamentous appendages, such as flagella, pili and fimbriae, in addition to EPS. It has

been reported that motility is critical for the initiation of E. coli biofilm formation (Van

Houdt and Michiels, 2005). Once the first microbial layer is formed, the subsequent

adhesion of other cells and abiotic material is favored (Melo, 2003).

With the substrate molecules reaching the cells inside the matrix, the production

of biomass and extracellular polymers leads to increased dry mass and thickness of

biofilm. Complex architectures with pedestal-like structures, water channels and pores

are formed to enable the convective and diffusive transport of oxygen and nutrients into

the biofilm (Melo, 2003; Teodósio et al., 2010).

The environment around the biofilm is responsible for cell dispersion from the

biofilm. Abrasion, erosion and sloughing processes can occur. Abrasion corresponds to

the scrap of biofilm by suspended particles, while erosion is the continuous removal of

single cells or small biofilm fragments caused by liquid shear. Sloughing is the

incidental loss of large particles of biomass from the biofilm (usually daughter cells)

because of the depletion of nutrients or dissolved oxygen at the biofilm base, or a

sudden increase of nutrient concentration in the bulk liquid (Gjaltema, 1996; Donlan,

2002). It is believed that these cells migrate to a new surface and form new biofilms.


- 10 -

In the present work, biofilms were formed by only one organism, although in

natural environments different organisms with different nutrient needs can create a

biofilm. The biofilm formed by mixed species are often thicker and more stable than

pure biofilm since there is a higher adaptation capacity to the medium (Kumar and

Anand, 1998).

2.4 Parameters involved in the biofilm life cycle

Biofilm establishment and development are dynamic and complex processes that

are strongly influenced by environmental conditions, cellular metabolism and genetic

control. In most cases, attachment will occur readily on surfaces that are rougher,

hydrophobic, nonpolar and coated by surface conditioning films (Pereira, 2001; Donlan,

2002). An increase in flow velocity, water temperature or nutrient concentration may

also increase the rate of microbial attachment, if these factors do not exceed critical

levels (Pereira, 2001). Properties of the cell surface, particularly the presence of

extracellular appendages, the interactions involved in cell-to-cell communication and

the production of EPS may possibly provide a competitive advantage for one organism

where a mixed community is present (Simões, 2005). Table 1 summarizes the main

variables involved in cell attachment and biofilm formation.

Table 1. Important variables in cell attachment and biofilm formation (based on (Donlan, 2002))

Properties of the adhesion surface Properties of the bulk liquid Properties of the cell

Texture or roughness Flow velocity Cell surface hydrophobicity

Hydrophobicity Nutrient availability Extracellular appendages

Conditioning film pH Signaling molecules

Temperature

On the other hand, mature biofilms are the result of cellular adaptations and

growth cycles determined by the nutrient diffusion and flow-dynamic conditions

(Teodósio et al., 2010a).


- 11 -

The hydrodynamics of the aqueous medium and the nutrient levels will be

considered in detail since they were the factors studied in this project.

2.4.1 Hydrodynamics

Biofilms in different environments are subjected to a very wide range of

hydrodynamic conditions (Sutherland, 2001). They have impact on biofilm formation in

terms of nutrients and oxygen supply and influence shear forces, and thus the capacity

of cells to attach to surfaces.

Shear force has been considered as one of the most important factors in the

formation of biofilms when the liquid flows at high velocities (high Reynolds numbers,

usually in the turbulent flow regime) over the biofilm surface (Vieira et al., 1993; Liu

and Tay, 2002; Melo and Flemming, 2010). There is evidence that shear force has

influences on the structure, mass transfer, production of exopolysaccharides and

metabolic/genetic behaviors of biofilms (Liu and Tay, 2002). As such, higher shear

stresses results in a thinner, denser and stronger biofilm (Rochex et al., 2008). The high

turbulence can cause two phenomena of opposite nature: it favors the transport of

nutrients to the surface, contributing to the growth and replication of cells in the

microbial layer and to the production of exopolymers; on the other hand, with

increasing flow velocity the shear stress forces also increase and that can cause further

erosion and detachment of biofilm portions, and then decrease the amount of biomass

attached to the solid support (Vieira et al., 1993; Percival et al., 1999; Pereira, 2001).

However, the reduction in biofilm biomass originates thinner biofilms, which could

benefit the transport of nutrients within the biofilm.

2.4.2 Nutrient availability

Several papers have been published during the last years concerning the effect of

nutrient levels on the formation and behavior of biofilm. The first studies observed that

high concentrations of nutrients in drinking water distribution systems increased the

number of cells in biofilms (Volk and LeChevallier, 1999; Frias et al., 2001). Work

carried out in a paper mill water stream also revealed that by increasing nutrient levels

(nitrogen and phosphorous), the biofilm amount also increased (Klahre and Flemming,


- 12 -

2000). The importance of this parameter was furthermore underlined by the conclusions

that maintaining low levels of nutrients is an effective way of controlling regrowth in

the system (van der Kooji, 1992).

Most of the work has been done by independent groups with different members of

Pseudomonas species. For instance, for Pseudomonas aeruginosa it is known that an

increase in nutrient concentration promotes biofilm formation (Peyton, 1996) and that

starvation leads to detachment (Delaquis et al., 1989; Hunt et al., 2004). For

Pseudomonas putida, Rochex and Lebeault (2007) observed an increase in biofilm

thickness when increasing glucose concentration up to a certain limit (0.5 g.L-1), above

which an additional increase of substrate reduced the biofilm accumulation rate as a

consequence of a higher detachment. It has also been reported for Pseudomonas species

that an increase in flow velocity or in nutrient concentration is associated with an

increase of cell attachment (Simões et al., 2010b).

Despite the lack of information on E. coli biofilms, contradictory results have

been reported. Dewanti and Wong (1995) found that biofilms developed faster when E.

coli O157:H7 was grown in low nutrient media. Later Jackson et al. (2002) noted that

the addition of glucose to media inhibited E. coli biofilm formation, an effect that may

be due to the classical repression system, i.e., cyclic AMP (cAMP) and cAMP receptor

protein (CRP) in E. coli. Eboigbodin et al. (2007) findings revealed that the relative

presence of glucose in the media, at the beginning of the growth phase, limits

aggregation in E. coli MG1655 by altering the concentration of functional groups from

macromolecules present on the bacterial surface. It has also been shown that the

presence of starved, stationary-phase like zones is important for biofilm formation (Ito

et al., 2008). On the other hand, other authors demonstrated that the total yield of E. coli

B54 (ATCC) growing in a biofilm increased linearly with increase of glucose up to 10

mmol.L-1 (Bühler et al., 1998), indicating that higher glucose concentrations may be

beneficial.

The glucose concentration may also not affect the biofilm formation, as verified

by Bagge et al. (2001) and Kim and Frank (1995) for Shewanella putrefaciens and

Listeria monocytogenes, respectively.

Although there is some information about the effect of glucose levels on biofilm

formation, little is known about the effect of varying nitrogen concentrations in the


- 13 -

same process. In biofilm reactors for ethanol production, low nitrogen media

encouraged the growth of yeast cells on plastic composite supports (Demirci et al.,

1997). However, in trickle-bed reactors for biological waste gas treatment, biofilm

growth seems to respond strongly to the amount of available nitrogen (Holubar et al.,

1999). A similar behavior was observed for Pseudomonas putida strain isolated from a

paper machine; the rate and extent of biofilm accumulation increased with nitrogen

concentration (from carbon/nitrogen=90 to carbon/nitrogen=20) (Rochex and Lebeault,

2007). Additionally, it is known that when the carbon/nitrogen ratio on the nutrient

supply is increased the polysaccharide/protein ratio is also increased (Huang et al.,

1994). Delaquis et al. (1989) showed that the depletion of nitrogen led to the active

detachment of cells from the Pseudomonas fluorescens biofilm, similarly to what

was observed under glucose limitation.

2.4.3 Hydrodynamic versus nutrient effects

The hydrodynamic conditions and the nutrients are the two main parameters that

influence biofilm growth, in particular the structure, density and thickness (Horn et al.,

2001). There is an interesting study that addresses the influence of hydrodynamics and

nutrients on biofilm structure (Stoodley et al., 1998). Stoodley et al. (1998) constructed

a diagram to predict biofilm morphotypes based on observational and theoretical

considerations of the relative influences of both parameters (Figure 4). At higher flow

velocity, where the influence of drag is high but mass transfer limitations are low, we

might expect drag reducing planar structures, the thickness of which depends on

nutrient concentration. However, at low shear, where the influence of mass transfer

limitations is high but drag is low, we might expect highly porous structures.


- 14 -

Figure 4. Two-dimensional nutrient concentration-flow velocity habitat domain diagram based on

observational and hypothetical considerations of mass transfer and shear on biofilm morphotypes

(Stoodley et al., 1998).

2.5 Platforms for in vitro biofilm studies

Intensive studies on the mechanisms of biofilm formation and resistance have

prompted the development of in vitro platforms for biofilm formation. These platforms

consist of models/systems of artificial biofilms that are easy to control and reproducible,

allowing a more detailed study of this phenomena. In vitro biofilm formation systems

range from static mono-cultures formed on membranes to mixed culture biofilms

growing under dynamic conditions. The focus of this thesis is on microtiter plates

(MTPs) but other common platforms, including flow cell systems, will also be

presented.

2.5.1 Microtiter plates

Considering bacterial growth in a laboratorial scale, several bioreactor designs

exist conferring different properties. Small-scale bioreactors have been widely preferred

because of their advantages of parallelization, automatization and cost reduction for

medium constituents especially in studies employing isotopically labeled tracer

substrates or substrates for mammalian cells (Kumar et al., 2004). In biotechnology,


- 15 -

preference is given to shaken bioreactors, including shake-flasks, test-tubes and

microtiter plates.

The concept of microtiter plates was first introduced in 1951 mainly for analytical

purposes (Manns, 2003). A microtiter plate (microplate or microwell plate) is a flat

plate with multiple wells used as small test tubes. A microplate typically has 6, 12, 24,

48, 96 or 384 wells arranged in a 2:3 rectangular matrix. The bottoms of the wells are

round or flat in shape and the wells are deep or shallow. The typical culture volume

used in microtiter plate varies from 25 µL to 5 mL (Kumar et al., 2004), depending on

the number of wells. Microtiter plates are manufactured in a variety of materials, being

the most common transparent polystyrene (PS), used for the measurement of the

microtiter plate absorbance (Teodósio et al., 2011b). PS microtiter plates are regularly

used as a model surface for adhesion and biofilm formation under laboratorial

conditions. Polystyrene has physico-chemical surface properties (hydrophobicity)

similar to those of other materials used in water distribution systems (Simões et al.,

2007). Illustrative photography of PS microtiter plates with 6, 48 and 96 wells can be

observed in Figure 5. Microplates may also be white pigmented, used in luminescence

assays, or black, used in fluorescence tests (Teodósio et al., 2011b). MTP-based systems

are closed (batch reactor-like) systems, in which there is no flow into or out of the

reactor during the experiment. As a result, the environment in the well will change (e.g.

nutrients become depleted, toxic products accumulate, etc.), unless the fluid is regularly

replaced (Coenye and Nelis, 2010).

Microtiter plates have been widely used for cell culture, tissue culture, enzyme

linked immunosorbent assay (ELISA) tests and high-throughput screening for

secondary metabolites (new drugs and antibiotics) and mutants (Hertzberg and Pope,

2000; Duetz, 2007). As biofilm model systems, microplates have been used to screen

for antimicrobial and anti-biofilm effects of various antibiotics, disinfectants, chemicals

and plant extracts (Amorena et al., 1999; Pitts et al., 2003; Shakeri et al., 2007; Quave

et al., 2008), to study microorganisms adhesion (Simões et al., 2010a) and to quantify

biofilm inhibition (Korenová et al., 2008). Since microplate assays are conducted in low

shear stress conditions, MTPs are good platforms to mimic the flow and biofilm

formation in urinary catheters or in arteries and veins. This system also allows

researchers to easily vary multiple parameters, including the composition of growth


- 16 -

media, incubation temperatures, presence or absence of shear stress and O2 and CO2

concentrations (Coenye and Nelis, 2010). A review of the most recent applications of

microplates in the biofilm study will be presented later.

Figure 5. Illustrative photograph of polystyrene microtiter plates used on biofilm formation: a) 6-well

microplate, b) 48-well microplate and c) 96-well microplate.

Microtiter plates offer the possibility of providing a large number of parallel and

miniaturize reactors with identical geometry and fluid dynamics (Kumar et al., 2004) in

a small space. Additionally, these devices are easy to handle with the use of multi-

channel micropipettes, pipetting robots, microplate readers and autosamplers (Duetz,

2007). Another advantage is that microtiter plate-based assays are fairly rapid and cheap

as only small volumes of reagents are required (Coenye and Nelis, 2010). In spite of

showing very promising characteristics, experiments performed using microtiter plates

and the colorimetric assays to assess biofilm formation can suffer from lack of

reproducibility between different laboratories, possibly due to the washing steps that are

researcher-dependent and to the existence of several protocol versions (Azevedo et al.,

2009). Furthermore, there is a risk of cross-contamination and excessive evaporation of


- 17 -

the growth media (Duetz and Witholt, 2001), as well as an assessment of biofilm

structure by microscopy has been difficulty due to the narrow geometry of the well

plates (Azevedo et al., 2009).

To overcome some of these problems, Ceri et al. (1999) developed a variation of

the traditional MTP model system. The Calgary Biofilm Device consists of a

polystyrene lid with 96 pegs that can be fit into a standard 96-well microtiter plate,

introducing an extra surface in the wells where the biofilm is to be formed and analyzed

(Figure 22 in Annex B). This device is not prone to contamination and leakage, and it is

more amenable to microscopic observation and control measurements (Azevedo et al.,

2009; Teodósio et al., 2011b). Recently, a "well plate microfluidic" device that allows

high-throughput screening of continuous flow biofilms was described (Benoit et al.,

2010). This dynamic system consists of microchannels integrated into a microplate,

where a pneumatic pump pushes fresh medium through the microchannel (containing

the biofilm) from an inlet well to an outlet well (containing spent medium) (Figure 23 in

Annex B).

2.5.1.1 Recent applications

Table 2 presents selected studies that have been made with biofilms in microtiter

plates in recent years. Although several methods have been used to study the formation

or removal of biofilms, the crystal violet (CV) staining is by far the most popular. The

growth of biofilms occurs mostly in polystyrene plates during 24 hours at 30 ºC or 37

ºC, depending on the microorganism. Shaking is rarely used and usually only the time

and temperature of incubation are indicated.

Table 2. Examples of recent applications of microtiter plates in the biofilms field

Microorganism Goal Method Experimental conditions Reference

Pseudomonas aeruginosa Screen for

antimicrobial activity

CV 24 h; 37 ºC;

96-well PS plate (Abdi-Ali et al.,

2006)

Candida albicans Screen for

antifungal activity

CFU Fluorescein

diacetate

48 h; 37 ºC; 100 rpm; flat-bottomed plate

(LaFleur et al., 2006)


- 18 -


Bacillus cereus Screen for


CV 72 h; 30 ºC;

96-well PVC plate (Auger et al., 2006)

Sinorhizobium meliloti Effect of nutritional and environmental

conditions CV

24 h; 30 ºC; n.s.; 96-well PVC plate

(Rinaudi et al., 2006)

Pseudomonas aeruginosa Effect of shear stress

and altered physiology

CV 16 h; 37 ºC; 250 rpm (o.s.) (Fonseca and Sousa, 2007)

Candida albicans Screen for antifungal activity XTT

24 h; 37 ºC; flat-bottomed PS 96-well

plate

(Ramage et al., 2007)

Escherichia coli Screen for biofilm-forming ability CV

16 h; 37 ºC; n.s.; flat-bottomed PS 24-well

plate

(Ferrières et al., 2007)

Acinetobacter sp2 Screen for


CV 24 h; 30 ºC; 8-well plate

(Shakeri et al., 2007)

Listeria monocytogenes Effect of flagellar motility

OD CV

1-5 days; 30 ºC; n.s.; 96-well PS plate

(Lemon et al., 2007)

Staphylococcus aureus Screen for biofilm-forming ability CV


plate (Rice et al., 2007)

Pseudomonas aeruginosa Screen for biofilm-forming ability CV

24 h; 30 ºC; PS plate

(Yang et al., 2007)

Saccharomyces cerevisiae Screen for biofilm-forming ability CV

0-4 h; 30 ºC; 200 rpm; 96-well PS plate

(Purevdorj-Gage et al., 2007)


24 h; 37 ºC; n.s.; PVC plate

(Bazire et al., 2007)

Burkholderia cenocepacia Screen for


Resazurin 24 h;

round-bottomed PS 96-well plate

(Peeters et al., 2008b)

Candida spp. Screen for antifungal activity XTT


plate (Pierce et al., 2008)



CV 20 h; 37 ºC; 96-well plate

(Overhage et al., 2008)



CV 24 h; 37 ºC, n.s.;

96-well PVC plate (Richards et al.,

2008)

Escherichia coli Effect of type 3 fimbriae CV

24 h; 37 ºC; 96-well PVC plate

(Ong et al., 2008)

Staphylococcus aureus Screen for


Resazurin 18 h; 37 ºC; 200 rpm;

flat-bottomed PS 96-well plate

(Sandberg et al., 2008)

Escherichia coli Screen for biofilm-forming ability

OD CV

24 h; 37 ºC; s.; PS

(Belik et al., 2008)


- 19 -


Listeria monocytogenes Screen for


CV XTT

6 h; 37 ºC (Sandasi et al., 2008)

Candida albicans Effect of growth medium XTT


plate

(Uppuluri et al., 2009)

Escherichia coli Screen for


CV Live/Dead


plate (Hou et al., 2009)

Staphylococcus epidermidis Screen for


CV 24 h; 37 ºC; n.s.;


(Hajdu et al., 2009)



CV 24 h; 37 ºC;


(Jagani et al., 2009)



Resazurin XTT

20 h; 37 ºC; n.s. flat-bottomed PS

(Pettit et al., 2009)

Escherichia coli Effect of type 1

fimbriae and mannose

CV 24-72 h; 37 ºC; n.s.;


(Rodrigues and Elimelech, 2009)

Escherichia coli Screen for mutants CV 24 h; 30 ºC; n.s.;


(Puttamreddy et al., 2010)

Cryptococcus neoformans Screen for


XTT 48 h; 37 ºC; n.s.; 96-well PS plate

(Martinez et al., 2010)

Escherichia coli Screen for


CV 24 h; 37 ºC; n.s.;

96-well plate (Choi et al., 2010)

Helicobacter pylori Screen for biofilm-forming ability CV

72 h; 37 ºC; 80-150 rpm; 12-well plate with glass

slides

(Yonezawa et al., 2010)

Drinking water-isolated bacteria

Screen for biofilm-forming ability CV

24-72 h; RT, 150 rpm; flat-bottomed PS 96-well

plate

(Simões et al., 2010a)

Stenotrophomonas maltophilia

Screen for biofilm-forming ability CV


plate

(Pompilio et al., 2010)



Safranin 24 h; 37 ºC;

96-well PS plate (Son et al., 2010)

Listeria monocytogenes Screen for biofilm-forming ability

OD CV

Ruthenium red

48 h; 4-37 ºC; n.s.; flat-bottomed PS 96-well

plate

(Zameer et al., 2010)

Streptococcus mutans Screen for


CV 48 h; 37 ºC;

flat-bottomed 96-well plate (Daglia et al., 2010)


- 20 -


Actinobacillus pleuropneumoniae

Effect of growth conditions CV

6-24 h; 37 ºC; 96-well plate

(Labrie et al., 2010)

Candida albicans Screen for biofilm-forming ability XTT

66 h; 37 ºC; 75 rpm; flat-bottomed PS 96-well

plate (Noumi et al., 2010)

Candida albicans Screen for


XTT 48 h; 30 ºC;


(Ramage et al., 2011)

Staphylococcus aureus Screen for biofilm-forming ability CV

18 h; 37 ºC; round-bottomed PS 96-well

plate

(Delgado et al., 2011)


18 h; 35 ºC; n.s. 96-well plate

(Perez et al., 2011)

n.s., no shaking; o.s., orbital shaking; s., shaking; RT, room temperature; CFU, colony-forming unit.

2.5.1.2 Effect of orbital shaking on biofilm formation

To achieve a highly effective mixing in microtiter plates, it is essential to supply

enough energy for generating a macroscopic flow in the fluid. Several established

methods for creating mixing effects in microplates are shown in Figure 21 (Annex A).

We concentrated our study on horizontal orbital shaking because it is undoubtedly a

simple, cheap and non invasive way for mixing of assay components and improve the

aeration rates in microtiter plates (Duetz and Witholt, 2001; BioShake.com, 2010).

As any other bioreactor that is used for the cultivation of microorganisms, care

has to be taken to operate MTPs at suitable conditions. Characterization of oxygen-

transfer rates (OTRs) and degrees of mixing were performed by different authors. As a

consequence, the key operating variables in shaken microplate fermentations were

identified as shaking pattern (orbital or linear), shaking frequency and amplitude,

microwell diameter, liquid fill volume, and fluid properties such as diffusivity, density,

viscosity, and surface tension (Doig et al., 2005). In this thesis, the effect of shaking

amplitude (or diameter) will be studied.

Duetz and Witholt (2001; 2004) were the first to show the impact of the shaking

diameter on culture aeration. At the same shaking frequency (300 rpm) and working

volume (260 µL), a shaking diameter of 50 mm resulted in a threefold higher oxygen


- 21 -

transfer rate (OTR) in comparison to a shaking diameter of 25 mm (round deepwells

with 6.6 mm of internal diameter) (Duetz and Witholt, 2004). This large difference may

be partially explained by a larger mass transfer area inside the wells at a shaking

diameter of 50 mm (Duetz et al., 2000). Moreover, it can be due to a better degree of

vertical mixing caused by the amplitude in question. Actually, the shaking pattern

photos (Figure 6) show that the angle of the aqueous surface with the horizontal plane

was higher at a shaking diameter of 50 mm (which can be attributed to the higher

centrifugal force) and small waves occurred at the surface (Duetz and Witholt, 2001).

For 96-low-well microtiter plates and a culture volume of 200 µL, Herman et al. (2003)

also found a strong influence of the shaking diameter in the oxygen transfer. Similar

OTRs at smaller shaking amplitudes were only reached at much higher frequencies. By

computational fluid dynamics (CFD), it was also confirmed that the orbital shaking

diameter generally has a greater impact on liquid motion than the shaking frequency

(Hermann et al., 2003; Zhang et al., 2008).

Figure 6. Shaking pattern during orbital shaking at 300 rpm in a round microwell of 6.5 mm of a 96-low-

well MTP: a) at a shaking amplitude of 25 mm (OTR=16 mmol L-1 h-1; reasonable degree of mixing, non-

turbulent) and b) at a shaking amplitude of 50 mm (OTR=32 mmol L-1 h-1; good mixing up to the very

bottom, non-turbulent) (Enzyscreen).

2.5.1.3 Biofilm quantification

Several microtiter plate-based assays have been used to determine biofilm mass,

microbial cells in the biofilm and associated physiological activity, and extracellular

matrix (Table 3). Their final results are based either on absorbance or fluorescence


- 22 -

intensity at a certain wavelength, which means that fast and quantitative analyses are

obtained with a simple microplate reader (Azevedo et al., 2009). These assays also show

a broad applicability and a high repeatability for many microorganisms (Peeters et al.,

2008a).

Table 3. Microtiter plate-based assays (adapted from (Azevedo et al., 2009))

Characteristic Method References

Biofilm biomass Crystal violet (CV) assay (Stepanovic et al., 2000)

Microbial physiological activity Resazurin assay (Pettit et al., 2005)

XTT assay (Kuhn et al., 2003)

Fluorescein diacetate assay (Honraet et al., 2005)

Biofilm matrix Dimethyl methylene blue assay (Toté et al., 2008)

In the present work, two methods for the quantification of microbial biofilms

formed in 96-well microtiter plates were used: the crystal violet assay for biomass

determination and the resazurin assay for cell activity determination.

Crystal violet (CV) staining was first described by Christensen et al. (1985). CV is

a basic dye, which binds to negatively charged molecules surface and polysaccharides

in the extracellular matrix (Peeters et al., 2008a). Therefore, CV staining procedure

allowed us to visualize cells (both living and dead) that had attached to the well surface

as such cells are stained purple with crystal violet (Figure 7), whereas abiotic surfaces

are not stained (Stepanovic et al., 2000).

Figure 7. Illustrative photograph of CV assay applied to a 96-well microplate.


- 23 -

To assay the metabolic activity of adherent cells, resazurin (7-hydroxy-3H-

phenoxazin-3-one-10-oxide), also known as Alamar Blue, was used. Resazurin is a

nontoxic and water-soluble dye which is reduced by electron transfer reactions

associated with respiration, producing the highly fluorescent pink resorufin (Figure 8)

(O'Brien et al., 2000; Pettit et al., 2005). Upon reduction, resazurin changes color from

dark-blue to pink to clear, as oxygen becomes limiting within the medium. The first

stage of this reduction is not reversible and is due to the loss of an oxygen atom bound

to the nitrogen of the phenoxazine nucleus. The second phase of reduction to the

colorless stage is reversible by atmospheric oxygen (Mariscal et al., 2009).

Figure 8. Conversion of resazurin to resorufin by viable cells. The fluorescence produced is proportional

to the respiratory activity of cells (adapted from (Promega, 2011)).

2.5.2 Other common platforms

In contrast to MTP-based system, flow displacement systems are "open" systems

in which growth medium with nutrients is (semi-)continuously added and waste-

products are (semi-)continuously removed (Coenye and Nelis, 2010). Therefore, these


- 24 -

systems have the advantage of incorporating the important aspect of fluid flow in the

setup, allowing the simulation of biofouling that occurs for instance in industrial piping

and heat exchangers. Rotating annular reactors and flow cells are the main examples of

flow displacement systems with removable surfaces (Pereira, 2001).

Annular reactors are composed of two concentric cylinders, one static external

and one rotating internal on which a number of slides are mounted (Figure 24 in Annex

C). A motor drives the inner cylinder, providing liquid/surface shear. Coupons are flush

with the walls of the reactor and are therefore subjected to the same hydrodynamic

conditions. The reactor is usually operated in the same manner as a regular chemostat in

which nutrients are continuously being introduced at a desired flow rate and the effluent

is expelled by overflow. One great advantage of this reactor compared to the flow cells

is that the shear stress and the flow velocity are determined by the rotation speed of the

inner cylinder and thus independent of the feed flow rate (Gjaltema et al., 1994;

Teodósio et al., 2011b).

Flow cells have been used for more than 30 years for the study of dynamic

biofilms. Although they exist in a variety of shapes sizes, these systems can be divided

in two main classes: those that contain removable coupons and those that are

particularly well-suited for real-time non-destructive microscopic analyses of biofilms

(Teodósio et al., 2011b). Flow cells containing a large number of coupons (usually

between 8 and 20) will be referred as large-scale flow cells whereas those that are

primarily destined for microscopic observation will be designated by small-scale flow

cells.

The flow cells that are most suitable to the simulation of industrial biofilms often

have two very important features: (1) use of a large number of coupons or adhesion

surfaces for biofilm formation and (2) possibility of operation at high flow rates in

regimes of high turbulence and shear stress. Most of these large-scale flow cells are

based on the design introduced by Jim Robins and later modified by McCoy et al.

(1981), creating what is commonly described as the Modified Robbins Device. This

flow cell is basically a square channel pipe with coupons fixed to sampling plugs that

can be unscrewed from the walls. In order to simulate biofilm formation on a specific

surface, several materials like glass, silicone rubber, PVC and stainless steel can be used


- 25 -

to make the coupon. In our research group, a flow cell with half-pipe geometry was

coupled to a recirculating tank (Figure 25 in Annex C), given its similarity to the

circular section of the tubes found in industrial piping systems (Teodósio et al., 2011a).

This alternative configuration was used to achieve the high flow rates that are common

in industrial processes and study their effects on E. coli biofilm formation under

different nutrient conditions. Note that operation of large-scale flow cells is usually

more complicated and slow when compared to some of the other biofilm growing

systems but the wealth of information that can be extracted from these systems usually

pays off.

The most common geometries of small-scale flow cells are the flat plate and the

glass capillary flow cells. Both can be used with video capture systems to enable real-

time observation of microbial adhesion, division of biofilm cells and production of EPS.

Flat plate flow cells are usually made on a polycarbonate base plate (although anodized

aluminum is also used for increased mechanical resistance) and contain one or two

square or rectangular glass viewing ports. Some models include recesses to fit coupons

that can be constructed from different materials to simulate cell adhesion to different

surfaces. They are frequently restricted to low flow rates and to laminar flow

applications (Teodósio et al., 2011b). An illustrative photograph of a small-scale flow

cell can be observed in Figure 24 (Annex C).


- 27 -

3 MATERIALS AND METHODS

3.1 Microorganism

The microorganism used to produce biofilms was Escherichia coli JM109(DE3),

a Gram-negative bacterium commonly used as a host strain for recombinant protein

production. For long term storage, stocks of this strain were prepared on 30% glycerol

and stored at -80 ºC.

3.2 Growing the cellular culture and preparation of inocula

Material / Equipment:

Strain glycerol stock;

Media components (Merck KGaA): D(+)-glucose monohydrate, peptone from

meat (peptic) granulated (specifications in Annex D), yeast extract granulated

(specifications in Annex E), potassium dihydrogen phosphate (KH2PO4) and di-

sodium hydrogen phosphate (Na2HPO4);

Sodium chloride (NaCl) (Merck KGaA);

Distilled water;

Erlenmeyer flask;

15 mL FalconTM tubes (VWR);

Analytical balance (A&D);

Autoclave;

Orbital shaker;

Spectrophotomer (T80 UV/VIS Spectrometer; PG Instrument, Ltd);

Vortex mixer (Reax control, Heidolph);

Centrifuge (5810 R, Eppendorf).


- 28 -

Experimental procedure:

a) For cultivation of E. coli, a concentrated media was prepared: 5.5 g.L-1 glucose,

2.5 g.L-1 peptone, 1.25 g.L-1 yeast extract in phosphate buffer (1.88.g.L-1 KH2PO4

and 2.60 g.L-1 Na2HPO4), pH 7.0. The media was autoclaved at 121 ºC for 20 min.

b) A flask containing the previous liquid media (typically 200 mL of medium in a 1

L flask) was inoculated with 500 µL of a glycerol stock and incubated overnight in

an orbital shaker (120-150 rpm) at 30 ºC until the optical density OD610 reached 1.

! Caution: After thawing, the remaining glycerol stock must be discarded. The repeated

freezing and thawing of glycerol cultures may result in rapid loss of viability of the

stored bacterial cells.

c) Cells from the overnight grown cultures were harvested in 15 mL Falcon tubes

by centrifugation (4000 rpm for 10 min at 25 °C). The supernatants were discarded

and the cell pellets were resuspended in 10 mL of sterile saline solution (NaCl

0.85%).

Δ Critical step: Because E. coli cells tend to settle, the cell suspensions must be

vortexed vigorously after resuspending and before pipetting for the different

manipulations used in this and successive work steps.

d) Dilutions were performed for an optical density of 0.4 at 610 nm.

3.3 Biofilm formation system

3.3.1 Effect of individual variation of glucose concentration


Media components listed in 3.2;

Sterile and non-sterile distilled water;

50 mL FalconTM tubes (VWR);

50 mL volumetric flask;

96-well microtiter plates: polystyrene, flat-bottomed, = 6.9 mm, well volume

= 0.34 mL, growth area = 0.37 cm2 (Orange Scientific);


- 29 -

Petri plates: polystyrene, round, diameter = 90 mm (VWR);

Multichannel pipette (VWR);

Analytical balance (A&D);

Plate with heating and magnetic stirring;

Orbital shakers: 50 mm (CERTOMAT® BS-1, Sartorius AG) and 25 mm

(AGITORB 200, Abalab);

Cover clamp (CR1800, Enzyscreen);

Autoclave.


a) The basic media without glucose was prepared with 0.25 g.L-1 peptone, 0.125

g.L-1 yeast extract and phosphate buffer (0.188 g.L-1 KH2PO4 and 0.26 g.L-1

Na2HPO4).

b) Three media with different glucose concentrations – 0.25, 0.5 and 1 g.L-1 – were

prepared using the medium described above and 300 g.L-1 concentrated glucose

solution. Media were autoclaved at 121 ºC for 20 min and stored at 4 ºC during the

experimental time.

Δ Critical step: Since the glucose solution is very concentrated, it is necessary to

dissolve the glucose in about half of the final volume of solution (50 mL) by gentle

warming and stirring.

c) Two separate plates were prepared according with Figure 9 for each time

interval to allow the two assays (crystal violet staining and resazurin reduction) to

be performed concurrently. A total of 12 microtiter plates per experiment were used.

Six wells of each MTP were filled with 180 µL of each nutrient media and a total of

18 wells were inoculated with 20 µL of the standardized inoculum prepared in 3.2.

Controls included sterile water and the three nutrient media without cells.

! Caution: Between each column filled, two columns should be left empty to prevent

cross-contamination in case of spills. All manipulations of 96-well microtiter plates can

be performed on an open bench but proper microbiological aseptic techniques must be

followed to prevent contamination. The use of sterile Petri dishes and multichannel

pipettes is recommended for loading media into the wells.


- 30 -

d) Microtiter plates were incubated at 30 ºC for 60 h in two orbital shaking

diameters at the same frequency (150 rpm) and no shaking (0 rpm). Orbital shakers

with 50 and 25 mm diameter were used.

Figure 9. Microplate layout for testing the effect of glucose concentration.

3.3.2 Effect of individual variation of the peptone concentration

The material/equipment and experimental procedures described in 3.3.1 were

strictly followed to test the effect of individual variation of the peptone concentration,

with the exception of the following: the basic media, now without peptone, was

prepared with 0.55 g.L-1 glucose, 0.125 g.L-1 yeast extract and phosphate buffer (0.188

g.L-1 KH2PO4 and 0.26 g.L-1 Na2HPO4); three media with different peptone

concentrations – 0.25, 0.5 and 1 g.L-1 – were prepared using the basic media and 300

g.L-1 concentrated peptone solution.

3.3.3 Effect of individual variation of the YE concentration

The material/equipment and experimental procedures described in 3.3.1 were

strictly followed to test the effect of individual variation of the yeast extract

concentration, with the exception of the following: the basic media, now without YE,

was prepared with 0.55 g.L-1 glucose, 0. 25 g.L-1 peptone and phosphate buffer (0.188

g.L-1 KH2PO4 and 0.26 g.L-1 Na2HPO4); three media with different YE concentrations –


- 31 -

0.125, 0.5 and 1 g.L-1 – were prepared using the basic media and 300 g.L-1 concentrated

YE solution.

3.3.4 Effect of culture medium formulations

The material/equipment described in 3.3.1 was used to study the effect of different

culture medium formulations on biofilm formation.


a) Eight media formulations were prepared according to Table 4 and supplemented

with phosphate buffer (0.188 g.L-1 KH2PO4 and 0.26 g.L-1 Na2HPO4). They were

autoclaved at 121 ºC for 20 min and stored at 4 ºC during the experimental time.

Table 4. Media compositions for optimization tests

Culture media Glucose concentration

(g.L-1)

Peptone concentration

(g.L-1)

YE concentration

(g.L-1)

1 1.0 1.0 1.0

2 0.5 1.0 1.0

3 1.0 0.5 1.0

4 1.0 0.5 0.5

5 1.0 1.0 0.5

6 0.5 1.0 0.5

7 0.5 0.5 0.5

8 0.5 0.5 1.0

b) Two separate plates of each type (1 and 2) were prepared according with Figure

10 for each time interval to allow the two assays (crystal violet staining and

resazurin reduction) to be performed concurrently. Six wells of the sterile microtiter

plate were filled with 180 µL of each nutrient media and a total of 18 wells were

inoculated with 20 µL of the standardized inoculum prepared in 3.2. Controls

included sterile water and nutrient media without cells.


- 32 -

c) Microtiter plates were incubated at 30 ºC for 60 h in two orbital shaking

platforms (50 and 25 mm) at 150 rpm.

Figure 10. Microplate layout for optimization tests.

3.4 Biofilm monitoring and quantification

During each experience of 60 h, microplates were removed from the incubator after

every 12 h for biofilm quantification. The crystal violet assay and the resazurin assay

were applied to each pair of microtiter plates.


- 33 -

3.4.1 Crystal violet assay (CV assay)


Sterile and non-sterile distilled water;

99% ethanol;

Gram's crystal violet solution (Merck);

100% acetic acid (VWR);

Multichannel pipette (Eppendorf);

Microplate reader (SpectraMax M2E, Molecular Devices).


a) The microplate was inverted in a single quickly movement, discarding the

contents of the wells.

b) Each well was washed with 200 µL of sterile water to remove all non-adherent

cells and again emptied.

c) The remaining attached bacteria were fixed with 250 µL of ethanol per well, and

after 15 min, the content was removed by inverting the microtiter plate.

d) 200 µL of 1% (v/v) crystal violet was added and the biofilm was stained for 5

min at room temperature without shaking.

e) After the plate was again emptied, the dye bound to adherent cells was

resolubilized with 200 µL of 33% (v/v) acetic acid.

f) The absorbance was measured at 570 nm by using the microplate reader.

3.4.2 Resazurin assay


Sterile distilled water;

Resazurin (Sigma-Aldrich);

Multichannel pipette (VWR);



- 34 -


a) The microplate was inverted in a single quickly movement, discarding the

contents of the wells.

b) Each well was washed with 200 µL of sterile water to remove all non-adherent

cells.

c) 190 µL of fresh media (or sterile water) was added to correspondent wells after

rinsing followed by the addition of 10 µL of 0.1 g.L-1 resazurin solution.

d) The plate was immediately covered in aluminum foil and incubated in the dark

for 20 min at room temperature.

e) Fluorescence (λexcitation: 570 nm and λemission: 590 nm) was measured by using the

microplate reader.

! Caution: The resazurin solution is very light sensitive, so it should be covered with

aluminum foil during preparation and after that. It needs to be prepared with sterile

water to ensure it is only metabolized by live E. coli cells. For the same reasons, the

addition of resazurin to the plates should be done under sterile conditions and in

darkness.

Storage of the resazurin solution is not recommended for prolonged periods since the

activity of the reagent may decrease overtime.

3.5 Glucose quantification

Glucose quantification was performed by dinitrosalicylic colorimetric method

(DNS) on studies about the effect of glucose concentration variation and culture

medium optimization. This method was previously optimized and standardized for

smaller sample volumes (Teodósio et al., 2011a).


Distilled water;

Dinitrosalicylic acid (DNS) reagent;

Eppendorf tubes;


- 35 -

96-well microtiter plates: polystyrene, flat-bottomed, = 6.9 mm, well volume

= 0.34 mL, growth area = 0.37 cm2 (Orange Scientific);

Water bath;



a) Before resazurin assay, 25 µL of each tested medium (in duplicate) and 25 µL of

a well with water were stored in Eppendorf tubes at -20 ºC.

b) After thawing, 25 µL of DNS reagent was added to the tube containing the

sample (medium or water).

c) The tubes were dived in a water bath at 80 ºC for 5 min and then cooled in

frozen water.

d) When the tubes reached room temperature, 250 µL of distilled water was added

to each one.

e) 200 mL of the resulting reaction mixture was transferred to a well of a microtiter

plate and the absorbance was read at a wavelength of 540 nm.

f) A calibration curve was constructed using glucose standards within the

concentration range of 0.25 – 2.0 g.L-1 (Figure 27 in Annex F).

3.6 Statistical analysis

The average of at least four replicate wells was considered for each nutrient media

and independent experiment. Each mean absorbance or fluorescence value was

corrected by subtracting the absorbance or fluorescence reading of the respective

uninoculated control prior to statistical analysis.

All the results presented in Figure 11, 15 and 16-19 were obtained on triplicate

experiments for each condition, whereas Figure 13 and 14 included data from four

independent experiments. For the DNS method (Figure 12 and 20), two microtiter plate

wells of each nutrient media were used, and each experiment was performed thrice. The


- 36 -

error bars on the figures represent the standard deviation (SD) of the means of

experiments.


- 37 -

4 RESULTS AND DISCUSSION

Three different concentrations of glucose, peptone and yeast extract were

individually tested in order to assess the influence of the carbon and nitrogen sources on

the kinetics of E. coli JM109(DE3) biofilm formation. Simultaneously, we evaluated the

effect of the orbital shaking amplitude on biofilm development by this strain in

polystyrene flat-bottomed microtiter plates. In a final task, we tried to find a culture

media composition that would generate more biofilms in dynamic conditions, since a

dynamic model can better simulate some real-life environments. Biofilm growth was

monitored every 12 hours by crystal violet and resazurin assays.

4.1 Effect of individual variation of glucose, peptone and YE concentrations

Figures 11 and 12 show the results of the parameters followed in the glucose

study – biofilm mass (panels A), cell respiratory activity (panels B) and glucose

concentration in culture media (panels C) – for the three incubation conditions. Looking

at the crystal violet results (panels 1A, 2A and 3A), the general trend is that the amount

of E. coli biofilm formed increased with increasing glucose concentrations, as

previously demonstrated by Bühler et al. (1998). These results indicate that higher

glucose concentrations may be beneficial for biofilm formation, independently of the

hydrodynamic conditions in microtiter plates.

At a shaking diameter of 50 mm and in the first 24 hours, the biofilm in wells with

1 g.L-1 glucose exhibited greater growth in comparison to the biofilm formed in the

remaining media, but they took longer to enter the exponential phase (the highest

biofilm growth was between 12 to 24 hours). For this nutritional condition, higher shear

stresses appear to hamper the cell adhesion to the surface in the beginning. When the

biofilm reached a critical mass (at 12 hours for 0.5 g.L-1 of glucose and at 24 hours for 1

g.L-1 of glucose), it was possible to observe a large variation of the absorbance signals

for both glucose concentrations. Biofilm seems to have entered a state of dynamic

equilibrium as a consequence of the combined effect of hydrodynamics and glucose

levels; this was only evident for conditions of greater agitation (shaking diameter of 50

mm; 150 rpm). The cyclical biofilm maturation and subsequent dispersal pattern


- 38 -

probably occurred because it is no longer "profitable" for the bacterium to participate in

the biofilm. Probably, the bacteria composing the outermost layers of the biofilm

changed to the free-floating state (sloughing and erosion processes) while others died

due to the lack of nutrients, exposure to toxic metabolic wastes generated by the biofilm

and concentrated in the area, and liquid shear (Dunne, 2002). After dispersal of the

biofilm commences, more nutrients are available for stimulating growth (if this is not

the limiting factor), and it becomes favorable for the bacteria to aggregate and form a

biofilm again. This can explain the oscillatory behavior of the growth curve in panel

1A, which is identical to that represented in Figure 2 and found in the literature (Pereira,

2001; Melo and Flemming, 2010).

With an orbital shaking diameter of 25 mm (panel 2A), the curves for each

concentration of glucose appear sufficiently spaced and the highest absorbance values

were always obtained for the glucose concentration of 1 g.L-1. For the same cultivation

conditions (shaking diameter of 25 mm and 1 g.L-1 glucose), we observed that, after the

biofilm mass had reached the maximum at 24 hours, a slight decrease in the absorbance

values occurred until the end of experience. In this situation, the oscillatory behavior

was not noticed, so one may suspect that substrate and/or oxygen limitations were the

main factors forcing the microorganisms to detach. For the lower concentration media

(0.25 and 0.5 g.L-1), the detachment process did not occur; biofilms have probably

entered into a slow-growing or non-growing state.

Under static conditions, biofilm growth in media with 1 g.L-1 glucose was lower

during the initial phase (panel 3A of Figure 11). The peak of biofilm was again for the

time point of 24 hours, but the correspondent biomass was slightly lower than in

dynamic conditions. These results may be due to the single effect of natural convection

in the microplates. The steady state was only reached in wells with the most

concentrated media (1 g.L-1 glucose) after 24 hours. This implies that the attached cells

per unit surface area were constant until the end of experiment, although with periodic

fluctuations. Unexpectedly, for media containing 0.25 and 0.5 g.L-1 glucose, the amount

of biofilm tended to increase over time, reaching a maximum at 60 and 48 hours,

respectively. This could mean that E. coli biofilms take more time to establish

themselves simultaneously when the carbon content is low and there are no shear

stresses caused by orbital shaking. On the other hand, probably the remaining nutrients

in media can contribute to biofilm growth at the end of the experiment. Glucose was


- 39 -

not one of the available compounds since it was exhausted after 24 hours in the two

more diluted media, according to graph 3C of Figure 12. However, peptone and/or yeast

extract may have promoted the biofilm development after this period. In addition, shear

stresses promoting cell detachment did not exist in this case.

According to the literature review, most of the recent works including growth of

microbial biofilms in microtiter plates are performed in a 24-hour period (Table 2).

However, the results of static conditions show the value of monitoring the biofilm

development for longer periods in order to make a complete study of its kinetics when

subjected to different cultivation settings.

Panels 1C, 2C and 3C from Figure 12 include the results of the dinitrosalicylic

colorimetric method (DNS) used to follow the evolution of glucose content in culture

media. The highest glucose consumption occurred during the first 24 hours for all

media. Nevertheless, glucose has never been depleted after 60 hours for the highest

glucose concentration (1 g.L-1), which suggests that some type of limitation occurred,

such as oxygen limitation, other nutrient limitation or formation of toxic byproducts in

the E. coli fermentation process.


- 40 -

Figure 11. Experimental results for E. coli biofilm formed in three cultivation conditions (1 – d0=50 mm, 150 rpm; 2 – d0=25 mm, 150 rpm; 3 – no shaking) with different glucose

concentrations ( - 0.25 g.L-1, - 0.5 g.L-1 and - 1 g.L-1). A – Crystal violet assay (absorbance at 570 nm); B – Resazurin assay (fluorescence at λex: 570 nm and λem: 590 nm). Error

bars indicate standard deviations of three experiments.


- 41 -

Figure 12. Experimental results for E. coli biofilm formed in three cultivation conditions (1 – d0=50 mm, 150 rpm; 2 – d0=25 mm, 150 rpm; 3 – no shaking) with different glucose

concentrations ( - 0.25 g.L-1, - 0.5 g.L-1 and - 1 g.L-1). C – Glucose concentration (g.L-1). Error bars indicate standard deviations of three experiments.


- 42 -

The influence of meat peptone on E. coli biofilm formation in PS microplates was

determined by crystal violet (panels A from Figure 13) and resazurin (panels B from

Figure 13) assays for all cultivation conditions, as happened for glucose. According to

the product information (Annex D), meat peptones are proteins from animal sources that

have been broken down into amino acids and peptides to provide nitrogen for

microorganisms, inclusive some strains of Escherichia coli. Like glucose is the main

carbon source in our culture medium, peptone is the most important nitrogen source. Its

nitrogen content exceeds 13%.

Under higher shear forces, that is, when an orbital shaker with a diameter of 50

mm at 150 rpm was used (graph 1A), the biofilm amount was higher for the highest

peptone concentration (1 g.L-1). The maximum absorbance signal at 570 nm was

reached at 36 hours, and from this moment on the amount of biofilm within the richest

medium markedly decreased to the level of the remaining media. The depletion of

nitrogen probably led to the active detachment of cells from the E. coli biofilm,

similarly to what was found for P. fluorescens biofilm (Delaquis et al., 1989). In

contrast to what happened to the glucose under the same incubation conditions, the

multiple effect of hydrodynamic and nutritional composition was probably not so severe

in the peptone tests. Biofilm development was most likely essentially controlled by

nutrient availability in the microwells.

With the 25 mm-incubator (panel 2A) and without shaking (panel 3A), the growth

profiles for the different peptone concentrations were similar, showing that this nutrient

has greater impact on biofilm formation when shaking conditions are more intense.

Comparing both dynamic systems (panels 1A and 2A from Figure 13), it is clear

that more biofilm was obtained during orbital shaking at 50 mm amplitude; for this

orbital amplitude, the maximum absorbance signal was 0.207 ± 0.125, while for the

smaller diameter a maximum value of 0.145 ± 0.036 was detected. In fact, there is a

larger mass transfer area inside the wells at a shaking diameter of 50 mm due to the

deformation in the liquid surface (similar to the one shown in Figure 6). The relative

increase of the mass transfer area inside the wells is given by the formula ,

where is the Froude number (Duetz et al., 2000). In turn, this dimensionless number

represents the ratio between centrifugal and gravitational acceleration (Doig et al.,

2005):


- 43 -

(1)

where is the shaking diameter, is the shaking frequency and is the gravitational

constant. From the arc tangent of this number, the theoretical angle of the liquid inside

the wells can be calculated (Duetz et al., 2000). Table 5 shows the Froude number, the

relative increase of mass transfer and the theoretical angle of the aqueous surface with

the horizontal plane for the studied dynamic conditions.

Table 5. Effect of the shaking amplitude on mass transfer in microtiter plates

(mm) N (rpm) Fr Theoretical angle

25 150 0.3 1.0 45º

50 150 0.6 1.2 50º

Although we have not considered the OTR in experimental work, it is interesting

to predict the volumetric oxygen transfer coefficient, , in microplates when different

shaking amplitudes were used. Doig et al. (2005) modeled the in MTPs comprising

two separate correlations based on different dimensionless groups. The first describe the

increase in specific air-liquid surface area ( as a function of Froude and Bond

numbers and is represented as

(2)

for 96-well microplate, where is the final specific surface area and is the initial

specific surface area. The second correlation defines the Sherwood number in

terms of Reynolds and Schmidt numbers and is represented as

. An overall correlation for predicting the volumetric oxygen

transfer in round bottom plates becomes

D (3)


- 44 -

where is the diffusion coefficient. Using the correlation displayed in Equation 3, a

of 0.306 h-1 and 0.476 h-1 at 150 rpm was obtained for a shaking diameter of 25 and

50 mm, respectively (calculations are detailed in Annex G).

No theoretical or experimental value was found in literature for the

cultivation conditions used in this project. Nevertheless, it was possible to conclude

about the adequacy of the correlation presented in Equation 3 by comparing the

experimental and modeled oxygen transfer coefficients in similar incubation conditions

(namely, the same microplate type, filling volume and microwell vessel diameter). John

et al. (2003) measured a value of 130 h-1 in conventional 96-well round-bottomed

microtiter plates using an integrated optical sensor. A shaking amplitude of 1 mm and a

frequency of 1020 rpm were tested by the authors. If we consider the fluid properties

used in the previous calculations (Annex G), the empirical value for the measurement

with the oxygen sensor, 0.932 h-1, is three orders of magnitude lower. This discrepancy

allow us to state that the correlation defined by Doig et al. (2005) is not adequate to

predict the volumetric oxygen transfer coefficient for the orbital movements tested in

this work.

Looking at Table 5, the expected ratio between the mass transfer areas at 150 rpm

for shaking diameters of 50 and 25 mm is 1.2. This implies a higher oxygen transfer rate

and consequently a higher bacterial growth in microplates placed in the 50 mm-

incubator if oxygen becomes limiting. Furthermore, the higher biofilm growth may have

derived from a better degree of vertical mixing that provided higher nutrient levels to E.

coli cells, and thus promoted the bacterial growth within the biofilm. Last but not least,

it is known that higher shear levels are generated in the culture plates placed in the

orbital incubator of 50 mm: the higher the orbital amplitude, the higher the centripetal

force (Equation (1)). Possibly higher shear stresses existed in the surface where the

bacterium tried to attach and it developed strategies to withstand the adverse

hydrodynamic conditions on adhesion. E. coli can, under certain conditions, adhere

more strongly to colonizing surfaces with increasing shear forces due to the action of

the flagella or of lectin-like adhesion (Thomas et al., 2002). These interesting strategies

might explain how higher viable and total cell counts were obtained for higher shear

stresses on water (Percival et al., 1999).


- 45 -

Figure 13. Experimental results for E. coli biofilm formed in three cultivation conditions (1 – d0=50 mm, 150 rpm; 2 – d0=25 mm, 150 rpm; 3 – no shaking) with different peptone


bars indicate standard deviations of four experiments.


- 46 -

Figure 14 shows the influence of yeast extract concentration on biofilm mass

(panels A) and metabolic activity (panels B), under the tested hydrodynamic conditions.

Like peptone, YE is a complex, ill-defined mixture of natural origin that is often used as

a nutrient in bacterial cultures. It provides vitamins, nitrogen, amino acids and carbon in

microbiological and cell culture media. YE nitrogen content exceeds 10%, according to

the product information (Annex E).

In opposition to glucose and peptone, yeast extract does not seem to be a key

component for E. coli biofilm production in microtiter plates. There was no significant

difference in the amount of attached cells when varying the YE concentration in the

culture medium, even applying the highest shaking diameter.

In contrast to our expectations, the data shows that more biofilm was formed

under static conditions, around 36-60 hours for all yeast extract concentrations (0.125,

0.5 and 1 g.L-1) (panel 3A). Similar growth curves were obtained for glucose tests under

the same cultivation conditions (panel 3A from Figure 11). This result indicates that

probably there were nutrients at the end of the experiment contributing to biofilm

growth and no shear forces promoting cell detachment.


- 47 -

Figure 14. Experimental results for E. coli biofilm formed in three cultivation conditions (1 – d0=50 mm, 150 rpm; 2 – d0=25 mm, 150 rpm; 3 – no shaking) with different yeast extract


bars indicate standard deviations of four experiments.


- 48 -

Table 6 summarizes the maximum amounts of biofilm achieved with each nutrient

(glucose, peptone and yeast extract) under different shaking conditions. Comparing the

effects of the media compounds, we can conclude that glucose is the nutrient that

enables greater biofilm production by E. coli in microplates, followed by peptone and

finally by yeast extract, for which the lowest absorbance values were registered. This

summary table also shows that more biofilm was formed on the 50 mm-incubator,

except for yeast extract, reinforcing the positive impact of a higher shaking diameter on

biofilm formation. We can also see that, in most tested conditions, a 24 to 36 hour time

interval is long enough for this E. coli strain to reach the peak of biofilm mass.

Table 6. Maximum absorbance values of the CV method for individual variation of glucose, peptone and

yeast extract, and respective concentration and time point

Glucose Peptone Yeast extract

Incubation conditions Abs

C

(g.L-1) t

(h) Abs

C

(g.L-1) t

(h) Abs

C

(g.L-1) t

(h)

d0=50 mm; 150 rpm 0.265 ± 0.060 1 24 0.207 ± 0.125 1 36 0.121 ± 0.026 1 48

d0=25 mm; 150 rpm 0.259 ± 0.066 1 24 0.145 ± 0.036 0.5 24 0.136 ± 0.030 0.25 36

no shaking 0.225 ± 0.056 0.5 48 0.195 ± 0.060 0.25 24 0.193 ± 0.062 0.25 48

Abs, absorbance; C, concentration of the indicated nutrient; t, time at which the maximum value was obtained.

The reference culture media for this thesis consists of 0.55 g.L-1 glucose, 0.25 g.L-

1 peptone, 0.125 g.L-1 yeast extract and phosphate buffer (0.188 g.L-1 KH2PO4 and 0.26

g.L-1 Na2HPO4) (Teodósio et al., 2011a). Since the phosphate buffer is not a nutritional

element (just helps to maintain a constant pH), its effect on biofilm formation was not

considered. However, to ensure that the buffer does not contribute to biofilm growth,

some microwells were filled with 180 µL of phosphate buffer (0.188 g.L-1 KH2PO4 and

0.26 g.L-1 Na2HPO4) plus 20 µL of cell suspension, and the kinetics of biofilm

formation was also followed during 60 hours by crystal violet assay (data not shown). It

was noted that the raw absorbance signal (without subtraction of control) was constant

through time and significantly lower than the raw values obtained for glucose and

peptone tests. Comparing with yeast extract experiments, the raw absorbance values for

phosphate buffer were very similar, which confirms the low impact of yeast extract

concentration on E. coli biofilm formation in microtiter plates. Also some volume of the


- 49 -

phosphate buffer plus cells was plated in plate count agar (PCA) to control the

planktonic growth. As expected, there was no bacterial growth in buffer.

While crystal violet assay was performed to determine the total amount of biofilm

formed in microtiter plates, resazurin was used to measure the presence of active

biofilm bacteria. Panels B of Figure 11, 13 and 14 illustrate the results of the resazurin

technique.

In this work, the resazurin assay showed no added value for the quantification of

E. coli biofilms compared to the CV assay. Additionally the obtained fluorescence

values were very low when compared with those found in the literature (Peeters et al.,

2008a). Although resazurin microtiter assay is inexpensive, rapid and simple to

perform, one of its chief limitations is that it usually requires a relatively high number

of microorganisms to obtain reliable results (Mariscal et al., 2009). Probably the amount

of attached cells was not high enough and therefore the assay was conducted very close

to the detection limit. Also the fluorescence detected in the yeast extract experiments

was higher than in the other tests and with more associated error, particularly in the case

of the standing incubator (coefficients of variation were higher than 50%, even resulting

from four independent experiments). This can be a consequence of the instability of

resazurin solution.

4.2 Effect of culture medium formulations

After the study of the individual variation of glucose, peptone and yeast extract,

the next goal was to try to optimize the culture media composition described by

Teodósio et al. (2011a) for E. coli biofilm production. Eight combinations of the three

nutrients were experimented under dynamic conditions. It was decided to test only the

biofilm formation in shaken microplates because the agitation led to increased biofilm

development when varying individually each medium component. In addition, although

the majority of in vitro studies investigating detection of biofilm formation have been

performed under static conditions, the conditions met in vivo (i.e. fluid flow

over/through catheters, movements of artificial joints, etc.), are almost exclusively

dynamic (Stepanović et al., 2001). Interestingly, the individual tests indicated that, in


- 50 -

general, the amount of biofilm grown in microplates increased with increasing nutrient

concentrations in the broth. Thus, the two highest concentrations of glucose, peptone

and yeast extract, i.e. 0.5 and 1 g.L-1 were tested (Table 4) so that the culture medium

had the same nutrients of the original one. Phosphate buffer was added to maintain the

pH of the medium.

For the first time in this work, it was possible to see a small white spot in the

center of the well surface (Figure 15), simply by looking at the underside of the

microtiter plate. It is believed that this "white mass" corresponds to a biofilm or simply

to planktonic cells that for some reason (e.g. hydrodynamic stress, nutrient availability)

settle to the bottom of the wells. At a shaking diameter of 50 mm, the white spots began

to be visible by naked eye after 24 hours and they grew over time, so that after 48 hours

of incubation they had a larger diameter. Simultaneously, the medium of the wells

became more turbid compared to the medium without cells used as control, and the odor

of the microtiter plates at their opening was intense. Therefore, it can be concluded that

there was greater growth of cells than in the first phase of this work. With a shaking

platform of 25 mm of amplitude, the white spot was just visible at 48 hours at the

bottom of microwells. The visual inspection tells us that not only richest media seems to

lead to increased biomass growth, but also that the largest shear stress felt at an orbital

diameter of 50 mm appears to promote bacterial adhesion or deposition on the

polystyrene surface.

The question if these white spots are or not biofilms arose with the application of

the CV method. During the washing procedure, we observed the disruption and falling

off in pieces of the biomass layer. It resisted to the first inversion of the microplate and

washing with water, but most of the layer was removed with the addition of ethanol to

the wells (see the experimental procedure in Section 3.4.1). In microplates is difficult to

assess whether this biomass was only deposited planktonic bacteria or already

corresponded to the initial stage of biofilm formation, i.e. reversible attachment to the

pre-conditioned surface. 96-well microtiter plates have very narrow geometry and

would require advanced microscopy techniques for analysis. However, as "white mass"

has remained in the first two procedures, we consider that it was not a simple cell

deposition, but there were already forces responsible for cell adhesion and biofilm


- 51 -

formation. We believed that E. coli biofilms formed could not resist to physical and

chemical treatments (fixation with ethanol) because they were not cohesive enough.

Figure 15. Photograph illustrative of the "white mass" formed at the bottom of the microplate wells

(d0=50 mm; 48 hours). Thick red arrows point the mass attached/deposited in some wells. In medium

without cells (C) and water, it was not observed.

Figures 16 and 17 shows the evolution of biofilm mass over time for the two

cultivation conditions (d0=50 mm and d0=25 mm at 150 rpm). For an easier analysis, the

eight tested media were plotted on different graphs according to the glucose

concentration (on the left side media with 1 g.L-1 glucose and on the right media with

0.5 g.L-1 glucose).

The first impression one gets of both figures is that the medium 2 (0.5 g.L-1

glucose, 1 g.L-1 peptone and 1 g.L-1 yeast extract) is that one where the lower

absorbance signals at 570 nm were detected. It was not possible to find the optimal

C

C C

C

H2O


- 52 -

medium composition for the highest biofilm production, but three formulations showed

the highest values of absorbance, as it will be discussed.

Figure 16. Crystal violet results for E. coli biofilm amount in different media formulations (see the

detailed composition on Table 4) at 50 mm orbital shaking diameter and 150 rpm. The media plotted on

the left have 1 g.L-1 glucose, while those represented on the right side contain 0.5 g.L-1 glucose. Error bars

indicate standard deviations of three experiments.


detailed composition on Table 4) at 25 mm orbital shaking diameter and 150 rpm. The media plotted on

the left have 1 g.L-1 glucose, while those represented on the right side contain 0.5 g.L-1 glucose. Error bars

indicate standard deviations of three experiments.


- 53 -

Even though it is possible to draw some interesting conclusions about the media

optimization tests, the results of the crystal violet method do not reflect what was

verified by visual inspection. The absorbance values are actually very close to those

obtained in the individual tests of each nutrient, in which no biofilm was seen to the

naked eye. The maximum signal was also around 0.25. As said before, the initial steps

of the method might have led to the removal of parts of the biofilm and therefore

underestimated the amount of biofilm formed. Consequently, we cannot say that the

detected absorbance values are proportional to the mass of biofilm formed in

microwells; they resulted of the effect of two phenomena: the amount of developed

biofilm and its level of cohesion.

Comparing the two graphs for both shaking diameters (Figures 16 and 17), the

highest absorbance signals were obtained for the media with more glucose (media 2, 6,

7 and 8). The same type of graphs were constructed for comparison of peptone and

yeast extract concentrations (Figures 28 and 29 in Annex H), nevertheless it was not

possible to observe a clear effect of the single variation of these compounds on

absorbance values. Thus, glucose seems to have an important and potentiating role on

E. coli biofilm formation or cohesion. In other words, with higher levels of glucose,

microbial cells adhered more to the surface or released less as determined by the crystal

violet assay.

Looking at Figure 16, yeast extract is not at all an essential nutrient for biofilm

growth or cohesion in our in vitro platform. The kinetic curves of culture media 3 and 4

have a similar pattern at a shaking diameter of 50 mm and the only difference in broth

composition is the YE concentration. The same happens with media 1 and 5. Analyzing

the second panel of Figure 16, it is possible to distinguish two pairs of culture media

with analogous growth curves: media 7/8 with higher absorbance values and media 2/6

with lower signals. Within each pair of media, the only variation is the yeast extract

content, so this is another proof of the little or null effect of this nutrient on biofilm

development or cohesion. Apart from the YE concentration which seems to be less

important, peptone concentration is changed between each pair. Medium 8 (with 0.5

g.L-1 peptone) presented larger absorbance values than medium 2 (with 1 g.L-1 peptone).

Also if we analyze Figure 28 (Annex H) where culture media are grouped by


- 54 -

concentrations of peptone, absorbance seems to respond strongly to lower nitrogen

levels (0.5 g.L-1).

Figure 18 shows the impact of the combination of glucose and peptone in

alternating concentrations, i.e. 1 g.L-1 glucose and 0.5 g.L-1 peptone and vice versa. For

both incubation conditions, formulations yielding more biofilm or more cohesive

biofilm were those with more glucose and less peptone (media 3 and 4).

Figure 18. Effect of the combination of glucose and peptone in alternating concentrations: media 3 and 4

include 1 g.L-1 glucose and 0.5 g.L-1 peptone, while media 2 and 6 have 0.5 g.L-1 glucose and 1 g.L-1

peptone (see the detailed composition on Table 4). Individual test includes data from experiments of

individual peptone variation (Figure 13-2A); the medium contains 0.55 g.L-1 glucose, 1 g.L-1 peptone and

0.125 g.L-1 YE. Error bars indicate standard deviations of three experiments, except for the individual test

that correspond to standard deviations of four experiments.


- 55 -

For some tested formulations of the culture media, the absorbance detected in the

last experimental point (as well as the standard deviation) was much higher than the

values recorded throughout the experiment. In the particular case of medium 6, the

absorbance at 60 hours was even higher than the double of the other signals detected for

the same media over time. These unexpected results probably means that E. coli cells

adhered more to the surface or detached less when applying the CV method.

Having in mind all results, it is possible to conclude that in media 2 and 6 the

lowest signals were detected and in medium 4 (1 g.L-1 glucose, 0.5 g.L-1 peptone and 1

g.L-1 YE), medium 5 (1 g.L-1 glucose, 1 g.L-1 peptone and 0.5 g.L-1 YE) and medium 8

(0.5 g.L-1 glucose, 0.5 g.L-1 peptone and 1 g.L-1 YE) the higher absorbance signals were

measured (Figure 19).

Figure 19. The culture media where were detected the highest and lowest absorbance values under

dynamic conditions (see the detailed compositions on Table 4). Error bars indicate standard deviations of

three experiments.

The resazurin assay was also used in the optimization tests to detect active biofilm

cells (Figure 30 in Annex H). Although this technique not added much information, the

fluorescence values were higher when compared to those obtained in the individual tests


- 56 -

of media compounds. The maximum fluorescence values were between 300 and 350,

hence the adhered cells were more metabolically active.

Glucose concentration was followed through time in culture media by DNS

method. As shown in Figure 20, glucose was exhausted in the tested media after about

36 hours, even in those that contained 1 g.L-1 of glucose. Thus, we can now say that

there is no oxygen limitation that hinders the total depletion of glucose in the microplate

wells. Possibly the amount of glucose that was not consumed in the first phase of this

project (Figure 12) was now shifted to a higher bacterial growth, as confirmed by visual

inspection (Figure 15).

Figure 20. Glucose concentration in different media formulations under dynamic conditions (see the

detailed compositions on Table 4). Error bars indicate standard deviations of three experiments.

Although oxygen from ambient air diffuses constantly through the microtiter plate

material (PS) into the samples (Arain et al., 2006), the apparent drawback of microbial

growth in microtiter plates is its limited oxygen transfer capacity. Therefore, the use of

microplates for the growth and maintenance of microbial strains has been mainly

limited to Escherichia coli and yeasts. For this purpose, the oxygen limitation is


- 57 -

generally less relevant since E. coli and the majority of yeast can grow anaerobically

(Duetz et al., 2000).


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5 CONCLUSIONS AND PERSPECTIVES FOR FURTHER RESEARCH

From the work presented in this thesis some major conclusions can be drawn:

This is the first study about the effect of various nutrients as well as the orbital

shaking diameter on the ability of Escherichia coli JM109(DE3) to form

biofilms in microtiter plates. It was demonstrated that the composition of the

medium and hydrodynamics have significant impact on this biological event.

The individual variation of glucose content in media indicated that the amount

of E. coli biofilm increases with increasing glucose concentrations. Therefore,

higher glucose concentrations may be beneficial for biofilm formation under

tested conditions (d0=50 mm, 150 rpm; d0=25 mm, 150 rpm; no shaking).

Peptone has greater impact on biofilm development when shaking conditions

are more intense, i.e. under a larger orbital shaking diameter.

The variation of yeast extract concentration has no significant impact on the

amount of attached cells, in the range of concentrations and cultivation

conditions used.

Optimization tests seem to indicate that glucose is the parameter with greater

influence on the absorbance values obtained in the crystal violet method for

both dynamic conditions under investigation. This may be a consequence of the

higher amount of E. coli biofilms formed or the establishment of more

cohesive biofilms in microplates.

An optimal medium composition for a high biofilm production at laboratory

scale was not identified, but three formulations presented higher absorbance

values: medium 4 (1 g.L-1 glucose, 0.5 g.L-1 peptone and 1 g.L-1 YE), medium

5 (1 g.L-1 glucose, 1 g.L-1 peptone and 0.5 g.L-1 YE) and medium 8 (0.5 g.L-1

glucose, 0.5 g.L-1 peptone and 1 g.L-1 YE).

With the optimization experiments, it was possible for the first time to see a

small white spot in the center of the wells which is thought to be biofilm.


- 60 -

Increasing nutrient concentrations in the broth probably promote planktonic

and sessile growth in microwells and lead to the depletion of the carbon source.

Crystal violet and resazurin assays are simple, fast and appropriate for high-

throughput measurement of biofilms in a MTP. However, the resazurin did not

show added value on the quantification of E. coli biofilms compared to the CV

assay. The washing technique of the CV method needs to be optimized to avoid

false-negative results.

Dynamic conditions should be included as one of the key parameters in the

study of in vitro biofilm formation in microtiter plates. The results of

individual tests showed that the use of orbital incubators with a shaking

amplitude of 50 mm leads to higher shear stresses that positively influence the

adhesion of bacteria to the substrata.

The experimental system used (96-well microtiter plate) is suitable for the

evaluation of E. coli biofilm growth under different environmental conditions.

Throughout the experimental execution of this work, there was a need to collect

more information on the topic addressed in this dissertation. Therefore, the following

topics are proposed for short-term achievement:

Predict shear stress level in 96-well microtiter plates by numerical simulation.

The use of computational fluid dynamics software is particularly useful to

predict different flow variables, such as shear and normal stresses exerted on

the wells, which are highly relevant to understand the development and growth

of biofilms.

Optimize the washing protocol of the crystal violet assay for the cultivation

conditions already defined.

Washing of a biofilm is a step of the utmost importance since it is supposed to

remove all non-adherent cells and simultaneously provide the preservation of


- 61 -

biofilm integrity. It is clear that insufficient washing may lead to false-positive

results, while excessive may lead to false-negative results, as probably

occurred in the second part of this work.

Test the effect of a standardized culture media - Muller-Hinton - on biofilm

formation by E. coli JM109(DE3) in microtiter plates.

This culture medium is commonly used in our research group for the

determination of minimum biofilm inhibitory concentrations. For a different E.

coli strain (CECT 434), this rich medium (2 g.L-1 meat infusion, 17.5 g.L-1

casein hydrolysate and 1.5 g.L-1 starch) cause an intense growth of planktonic

and sessile cells.

Monitor the planktonic growth in microwells.

There are several ways to measure bacterial growth before the application of

CV and resazurin assays, but the easiest one is to measure the turbidity in wells

using a microtiter-plate reader. This was tested during this work but the method

needs further optimization to yield reproducible results.

Include more high-throughput methods to study the biofilm formation in

microtiter plates.

The XTT assay can be used in place of the resazurin method to determine the

metabolic activity of biofilm cells (Kuhn et al., 2003). It can be also interesting

to quantify the E. coli biofilm matrix using the dimethyl methylene blue dye

(Toté et al., 2008).

Validate the results of 96-well microtiter plates in flow cell reactors.

To work with high shear stress conditions, like those found in the food

industry, the culture media optimized in this project will be tested in a flow cell

system that has already been validated (Teodósio et al., 2011a) and in a second

one (at a smaller scale) that is being built.


- 62 -

The realization of some of this future work is enclosed in the work plan of my

application for a PhD grant submitted to the Portuguese Foundation for Science and

Technology.


- 63 -

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- A1 -

ANNEXES


- A2 -

ANNEX A: Methods for generating mixing effects in microtiter plates

Figure 21. Microplate mixing tools (BioShake.com, 2010).


- A3 -

ANNEX B: Variations of the traditional microtiter plate-based model system

Figure 22. Surface of Calgary Biofilm Device pegs with biofilm (Wei et al., 2006).

Figure 23. BioFlux high-throughput system for screening of flow biofilm viability and other parameters:

a) photograph of BioFlux system and b) schematic diagram showing the system operation (adapted from

(Benoit et al., 2010)).


- A4 -

ANNEX C: Commonly used flow displacement systems for biofilm studies

Figure 24. Schematic representation of a rotating annular reactor (Gjaltema et al., 1994).

Figure 25. A large-scale flow cell reactor: a) illustrative photograph and b) schematic representation of

the experimental apparatus system (adapted from (Teodósio et al., 2011a)).


- A5 -

Figure 26. Illustrative photograph of the experimental apparatus system used to perform biofilm

formation on a small-scale flow cell reactor.


- A6 -

ANNEX D: Specifications of Peptone from Meat (peptic), granulated (Merck

Microbiology Manual)


- A7 -

ANNEX E: Specifications of Yeast Extract, granulated (Merck Microbiology

Manual)


- A8 -

ANNEX F: Calibration curve of glucose

Figure 27. Glucose concentration standard curve (linear regression: y=0.4382x; R2=0.9949).

0.0

0.2

0.4

0.6

0.8

1.0

0.0 0.5 1.0 1.5 2.0 2.5

Ab

sorb

ance

(5

40

nm

)

Glucose concentration (g.L-1)


- A9 -

ANNEX G: Calculation of the volumetric oxygen transfer coefficient

Common variables:

We considered the physical and chemical properties of water at 30 °C. The Bond

number was obtained at a wetting tension (W) of 72.3 mN.m-1 (Doig et al., 2005).

Bond number:

Reynolds number:

Schmidt number:

Initial specific surface area:

For shaking amplitude of 25 mm:

Froude number:

For shaking amplitude of 50 mm:

Froude number:


- A10 -

ANNEX H: Extra graphics with results of culture media formulations


detailed compositions on Table 4) under dynamic conditions. The media plotted on the left have 1 g.L-1

peptone, while those represented on the right side have 0.5 g.L-1 peptone. Error bars indicate standard

deviations of three experiments.


- A11 -


detailed compositions on Table 4) under dynamic conditions. The media plotted on the left have 1 g.L-1

yeast extract, while those represented on the right side have 0.5 g.L-1 yeast extract. Error bars indicate

standard deviations of three experiments.


- A12 -

Figure 30. Resazurin results for E. coli biofilm formed under dynamic conditions in different media

formulations (see the detailed compositions on Table 4). Error bars indicate standard deviations of three

experiments.

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