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
When you make the finding yourself — even if you’re the last person on Earth to see the light — you’ll never forget it.
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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.
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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
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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 ... 11.1 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
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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).
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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. ... 41
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
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 four experiments. ... 45 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
concentrations ( - 0.125 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 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
Figure 17. Crystal violet results for E. coli biofilm amount in different media formulations (see 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
Figure 28. Crystal violet results for E. coli biofilm amount in different media formulations (see 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 Figure 29. Crystal violet results for E. coli biofilm amount in different media formulations (see 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)
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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 biofilmsBiofilms 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).
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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.
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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)).
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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.
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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).
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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,
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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
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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.
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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,
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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
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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
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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 antimicrobial Screen for
activity CV
24 h; 37 ºC;
96-well PS plate (Abdi-Ali et al., 2006)
Candida albicans antifungal activity Screen for Fluorescein CFU diacetate
48 h; 37 ºC; 100 rpm;
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Microorganism Goal Method Experimental conditions Reference Bacillus cereus antimicrobial Screen for
activity 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 antifungal activity Screen for XTT flat-bottomed PS 96-well 24 h; 37 ºC; plate
(Ramage et al., 2007)
Escherichia coli Screen for biofilm-forming ability CV flat-bottomed PS 24-well 16 h; 37 ºC; n.s.; plate (Ferrières et al., 2007) Acinetobacter sp2 Screen for antimicrobial activity 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 flat-bottomed PS 96-well 24 h; 37 ºC; n.s.;
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)
Pseudomonas aeruginosa Screen for biofilm-forming ability CV 24 h; 37 ºC; n.s.;
PVC plate (Bazire et al., 2007)
Burkholderia cenocepacia antimicrobial Screen for
activity Resazurin 24 h; round-bottomed PS 96-well plate (Peeters et al., 2008b)
Candida spp. antifungal activity Screen for XTT flat-bottomed PS 96-well 24 h; 37 ºC; n.s.;
plate (Pierce et al., 2008)
Pseudomonas aeruginosa antimicrobial Screen for
activity CV
20 h; 37 ºC;
96-well plate (Overhage et al., 2008)
Pseudomonas aeruginosa antimicrobial Screen for
activity 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 antimicrobial Screen for
activity 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.;
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Microorganism Goal Method Experimental conditions Reference Listeria monocytogenes antimicrobial Screen for
activity
CV
XTT 6 h; 37 ºC (Sandasi et al., 2008)
Candida albicans Effect of growth medium XTT flat-bottomed PS 96-well 24 h; 37 ºC; n.s.; plate
(Uppuluri et al., 2009)
Escherichia coli antimicrobial Screen for activity
CV Live/Dead
24 h; 37 ºC; flat-bottomed PS 96-well
plate (Hou et al., 2009)
Staphylococcus epidermidis antimicrobial Screen for
activity CV
24 h; 37 ºC; n.s.; flat-bottomed PS 96-well
plate (Hajdu et al., 2009)
Pseudomonas aeruginosa antimicrobial Screen for
activity CV
24 h; 37 ºC; flat-bottomed PS 96-well
plate (Jagani et al., 2009)
Staphylococcus aureus antimicrobial Screen for activity
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.; flat-bottomed PS 96-well plate (Rodrigues and Elimelech, 2009)
Escherichia coli Screen for mutants CV flat-bottomed PS 96-well 24 h; 30 ºC; n.s.; plate
(Puttamreddy et al., 2010)
Cryptococcus neoformans antimicrobial Screen for
activity XTT
48 h; 37 ºC; n.s.;
96-well PS plate (Martinez et al., 2010)
Escherichia coli antimicrobial Screen for
activity 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
24 h; 37 ºC; flat-bottomed PS 48-well
plate
(Pompilio et al., 2010)
Staphylococcus aureus antimicrobial Screen for
activity 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 antimicrobial Screen for
activity CV
48 h; 37 ºC;
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Microorganism Goal Method Experimental conditions Reference 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 flat-bottomed PS 96-well 66 h; 37 ºC; 75 rpm;
plate (Noumi et al., 2010)
Candida albicans antimicrobial Screen for
activity XTT 48 h; 30 ºC; flat-bottomed PS 96-well plate (Ramage et al., 2011)
Staphylococcus aureus Screen for biofilm-forming ability CV round-bottomed18 h; 37 ºC; PS 96-well plate
(Delgado et al., 2011)
Pseudomonas aeruginosa Screen for biofilm-forming ability CV 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
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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
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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).
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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
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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).
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3 MATERIALS AND METHODS
3.1 MicroorganismThe 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);
