MINI-REVIEW

“Biofilmology”: a multidisciplinary review of the studyof microbial biofilms

Esther Karunakaran & Joy Mukherjee &

Bharathi Ramalingam & Catherine A. Biggs

Received: 24 December 2010 /Revised: 26 March 2011 /Accepted: 27 March 2011 /Published online: 3 May 2011# Springer-Verlag 2011

Abstract The observation of biofilm formation is not a newphenomenon. The prevalence and significance of biofilm andaggregate formation in various processes have encouragedextensive research in this field for more than 40 years. In thisreview, we highlight techniques from different disciplines thathave been used to successfully describe the extracellular,surface and intracellular elements that are predominant inunderstanding biofilm formation. To reduce the complexitiesinvolved in studying biofilms, researchers in the past havegenerally taken a parts-based, disciplinary specific approach tounderstand the different components of biofilms in isolationfrom one another. Recently, a few studies have looked intocombining the different techniques to achieve a more holisticunderstanding of biofilms, yet this approach is still in itsinfancy. In order to attain a global understanding of theprocesses involved in the formation of biofilms and toformulate effective biofilm control strategies, researchers inthe next decade should recognise that the study of biofilms, i.e.biofilmology, has evolved into a discipline in its own right andthat mutual cooperation between the various disciplinestowards a multidisciplinary research vision is vital in this field.

Keywords Biofilms . Biofilmology . Extracellular .

Intracellular .Multidisciplinary . Surface

Introduction

In nature, bacteria can exist as a consortia adherent to eachother (aggregates) and/or to surfaces (biofilm) forming

sessile communities able to respond and adapt to changes inthe environment or perform highly specialised tasks similarto multi-cellular organisms (Costerton et al. 1999, O’Tooleet al. 2000; Hall-Stoodley and Stoodley 2002). Bacterialbiofilms, generally described as cells bound together byextracellular polymeric substances and attached to asurface, involve both cell–surface and cell–cell interactionsas part of the developmental process (Davey and O’Toole2000, O’Toole et al. 2000). Bacterial aggregation is thephenomenon by which microorganisms interact with eachother, forming a steady, multi-cellular cluster (Marshall1976). Hence, aggregates can also be described as biofilms,where the surface substratum is much less defined (Daveyand O’Toole 2000).

The ability of bacteria to form multi-cellular communitieseither attached to each other or to a surface is of greatimportance in a diverse range of disciplines and applicationsspanning biotechnological, environmental and medical indus-tries, which can have both a positive or a negative effect onmodern society. The formation of different aggregates withinwastewater treatment processes such as activated sludge flocs(Biggs and Lant 2000) or aerobic or anaerobic granules (Adavet al. 2008; Skiadas et al. 2003) are well-known positiveapplications of bacterial aggregation which enhance separationand treatment efficiencies. Also, the aggregation of yeastwithin the brewing process has a positive influence on theoverall process (Bauer et al. 2010). Biofilm formation is alsopositively exploited in bioremediation (Cohen 2002) andmicrobial fuel cell technology (Erable et al. 2010). In thesecases, successful formation of aggregates in suspension, orbacterial attachment to surfaces, results in enhanced efficiencyof the process. Conversely, however, bacterial aggregation andbiofilms can also have a detrimental effect on industrialprocess efficiency and public health, e.g. annual worldwideprocess engineering costs related to biofouling is in the excessof hundreds of millions of pounds (Riedewald and Sexton

E. Karunakaran : J. Mukherjee : B. Ramalingam :C. A. Biggs (*)Department of Chemical and Biological Engineering,ChELSI Institute, The University of Sheffield,Sheffield S1 3JD, UKe-mail: c.biggs@sheffield.ac.uk

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2006), and approximately 65% of hospital-acquired infectionsare related to surface-attached microbes (Resch et al. 2005).

The observation of biofilm formation is not a newphenomenon. The earliest reports of biofilms were madeabout 80 years ago (Henrici 1933, Zobell 1943). Theubiquity of biofilms in the environment was realised about40 years ago (Costerton 2007). Consequently, since theearly 1970s, interests in biofilm research and biofilm-basedtechnologies have steadily increased. Indeed a literaturesearch for “biofilm” specifically in the title of paperspublished from 2000 to 2010 resulted in 5,100 papers, witha steady increase year on year (see Fig. 1) (Web of Science(wok.mimas.ac.uk) Science Citation Index Expanded,accessed 15/03/11).

Several approaches have been applied to investigate thephenomenon of bacterial cell–cell and cell–surface interac-tions. In this review, we will highlight key techniques (e.g.microscopy, spectroscopy, genetics and proteomics) andfindings from various disciplines (e.g. surface and polymerscience, microbiology, biochemistry) to describe the extracel-lular, surface and intracellular components of biofilms. In sodoing, the crucial role of multidisciplinarity in advancingbiofilm research is demonstrated, promoting the creation of“biofilmology” as a discipline in its own right. Through anexamination of potential limitations in an exclusivist approachto biofilm research, this review also highlights the areas forfuture studies emphasising on the potential for furthermultidisciplinary research.

The extracellular environment is key: what is goingon outside?

The immediate environment of the cells within a biofilm isdensely populated with polymers, and extensive research

has been targeted towards understanding the interaction ofthese polymers within biofilms. The extracellular polymericsubstances (EPS) that form the matrix account for over 90%of the biofilm content and are chiefly microbial in origin(Flemming and Wingender 2010). EPS has been defined asan umbrella term for a group of poorly understood macro-molecules located outside the bacterial cell (Cooksey1992), although the presence of various components ofthe EPS, such as proteins, carbohydrates, DNA andmembrane vesicles, has been verified (Flemming et al.2007). The production of extracellular polymers is anexpensive metabolic investment; therefore, it can beinferred that the presence of EPS is crucial for biofilmformation and maintenance. The production of EPS bybacteria upon adhesion to surfaces has indeed been hailedas the “hallmark” of biofilm formation (Stoodley et al.2002); however, the mechanism of interaction between theEPS components resulting in a stable matrix is, to date, afertile field of research.

Extensive research undertaken in the past few decadeshas focused on understanding the adhesive and cohesiveproperties of these biopolymers. The analytical techniquesused for studying the EPS components can be broadlyclassified into two types: non-destructive techniques andtechniques that study the EPS extracted from disruptedbiofilms. Confocal laser scanning microscopy (CLSM) isthe most popular, non-destructive technique used (Neu etal. 2010) to monitor the time-resolved accumulation ofvarious EPS components within biofilms. In this technique,the different components of the EPS can be identifiedvisually by the addition of fluorescent probes. For instance,localisation of proteins using fluorescein isothiocyanatelabelling, polysaccharides with Calcofluor white or conca-navalin A labelling and nucleic acids using SYTOX Bluelabelling can be visualised using CLSM (for more details of

Fig. 1 Number of paperspublished in biofilm researchover the last 10 years

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the specific probes for each component, please refer to Adavet al. 2010). CLSM has played an important role in shapingour understanding of the spatial organisation and formationof micro-domains within biofilms (Lawrence et al. 2007).

Fourier-transformed infrared (FTIR) spectroscopy is an-other popular non-destructive technique for monitoring time-resolved EPS accumulation in biofilms. In this technique, theaccumulation of various EPS-associated functional groupsand conformational changes in the EPS polymers can bemonitored either by growing the biofilms directly on theattenuated total reflectance crystal (Delille et al. 2007; Quilèset al. 2010) or by growing biofilms on surfaces of interestlike stainless steel and plastics (Pink et al. 2005; Bosch et al.2006; Cheung et al. 2007; Ojeda et al. 2008). A microscopeattached to the FTIR (micro-FTIR) can also aid in theanalysis of micro-domains within biofilms, including theEPS matrix. Although greater detail regarding the spatialdistribution of EPS can be visualised from CLSM than fromreflectance micro-FTIR spectroscopy, still FTIR can provideuseful information about the functional groups in EPS thatplay an adhesive and cohesive role in the maintenance ofbiofilms (Geoghegan et al. 2008). FTIR spectroscopy of EPSextracted from disrupted biofilms has also been carried out(Eboigbodin and Biggs 2008; Tapia et al. 2009; Liang et al.2010; Karunakaran and Biggs 2011). The use of Ramanspectroscopy and surface-enhanced Raman spectroscopy, asdiscussed further in the next section, has also been exploredfor the analyses of EPS samples (Ivleva et al. 2009).

Although non-destructive, in situ monitoring techniquesare available, the more commonly employed strategy is toanalyse the EPS obtained by the inevitable disintegration ofbiofilms. Results from simple calorimetric assays for totalamount of proteins and carbohydrates and subsequentcalculation of protein–carbohydrate ratios suggest that,generally, a predominance of protein components rather thanpolysaccharides leads to the greater stability of flocs andbiofilms (Sheng et al. 2010). A detailed biochemical analysisreveals that the polysaccharide components can eithercontain homopolysaccharides like cellulose in Salmonellatyphimurium (Zogaj et al. 2001) or charged heteropolysac-charides that can either be polyanionic like in the case ofalginate in Pseudomonas aeruginosa (Evans and Linker1973; Wozniak et al. 2003) and colonic acid in Escherichiacoli (Danese et al. 2000) or polycationic in the case of theintercellular adhesin of Staphylococcus aureus (Götz 2002).A current understanding establishes that the interaction ofexopolymers with inorganic substituents like divalent cations(e.g. Ca2+ and Mg2+) and metal centres on surfaces serve tofurther influence the physical properties and enhance themechanical stability of flocs and biofilms (Biggs et al. 2001;Geoghegan et al. 2008; Körstgens et al. 2001).

A largely metabolic role is reserved for the extracellularproteins present within biofilms, and the predominance of

protein components in biofilms has led to the idea that the EPSmatrix could possibly function as an efficient external digestivesystem (Flemming and Wingender 2010). The biochemicalsignificance of the immobilisation of extracellular enzymeswithin the polysaccharide matrix has been verified in the caseof retention of extracellular lipase by alginate residues withinP. aeruginosa biofilms (Mayer et al. 1999). The active role ofenzymes like peptidases, polysaccharases and phosphataseshas been confirmed within biofilms and these enzymesincrease the bioavailability of nutrients in the surroundingenvironment (Romani et al. 2008; Neu and Lawrence 2009).

The interaction between the protein component and the EPSpolysaccharides can also be of structural significance as in thecase of the secreted TasA protein and exopolysaccharides inBacillus subtilis biofilms (Branda et al. 2006). Generally, theproduction of sugar-binding peptides, lectins, is also thoughtto contribute to the structural integrity of the biofilms (Neuand Lawrence 2009). A recent work on the detailedbiochemical analysis of the protein components of EPSattributes a small yet significant contribution of polycationicpeptides in maintaining the structural integrity of Bacilluscereus biofilms (Karunakaran and Biggs 2011).

The concept that the physical interaction between thepolymers in the matrix through electrostatic forces, van derWaals interaction, polar interactions and hydrogen bondinginfluences themechanical stability of the biofilm has also beendemonstrated by rotational viscosimetry (Mayer et al. 1999).The EPS molecules, by a process called polymer bridging,have also been found to play an important role inovercoming the electrostatic repulsion between the bacteriumand the surface, thus ensuring firm, irreversible attachment ofthe bacteria to the surface (Neu and Marshall 1990;Karunakaran and Biggs 2011). The outcome of such researchhas spurred investigations into novel surface coatings thatreduce bioadhesion (Yuan et al. 2009; Khoo et al. 2009).

In addition to physicochemical characterisation, the regu-lation of EPS production at the genetic level has been studiedin detail in several organisms. For instance, in B. subtilis, thematrix is composed of exopolysaccharides, produced bygenes encoded on a single operon (eps operon), and anextracellular protein, TasA (Kearns et al. 2005). The epsoperon and the genes involved in the production andprocessing of TasA have been demonstrated to be underthe control of several transcriptional regulators (Kearns et al.2005). The reduction of biofilm formation and maturationhas been confirmed visually in the appropriate deletionmutants (Kearns et al. 2005). Recently, the translationalcontrol of EPS production has also been shown to occur inB. subtilis (Irnov and Winkler 2010). Likewise, the control ofEPS production by the intracellular levels of cyclic-di-guanosine monophosphate is well studied (Jenal and Malone2006). An extensive analysis of the extracellular proteomesof the B. cereus group of organisms has revealed the role of

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the pleiotropic regulator, PlcR, in regulating extracellularprotein production (Gohar et al. 2002; Gohar et al. 2008;Oosthuizen et al. 2002).

Finally, both the desirable effects of biofilms such asenvironmental detoxification (Flemming and Wingender2010) and the undesirable effects of biofilms such asbiofouling of surfaces and insulation of the walls of heatexchangers (Flemming and Wingender 2001) are directlylinked to the sorptive properties of the matrix components.The stabilisation and concentration of extracellular enzymeswithin biofilms help the biofilms function as powerhouses ofdegradation of xenobiotics and organic polymers (Flemmingand Wingender 2010). On the other hand, the increased metalion binding by the metalloproteins within biofilms contributesto the biocorrosion of surfaces (Neu and Lawrence 2009).The colligative properties of the EPS polymers (Keiding et al.2001) insulate the walls of heat exchangers against convec-tive heat transfer and reduce the efficiency of industrialprocesses (Flemming and Wingender 2001).

Although investigations into the external environment ofbiofilms (e.g. EPS) have incorporated techniques from variousfields like microscopy, spectroscopy, biochemistry, surfacescience and genetics (Fig. 2) and have succeeded in providingthe research community with a wealth of information, a fewlimitations exist. The limitations arise due to the fact that noconsensus exists on the EPS extraction techniques and thecomplete recovery of all components of the EPS from abiofilm remains a challenge. The composition of the matrixpolymers can be easily perturbed by changes in cellularmetabolism and consequently can change the physical forces

that stabilise the biofilms. Therefore, research into extracellu-lar polymers when supplemented with an understanding of thenature of the physical interaction between the bacterial cellsurface and the exopolymers, coupled with an appreciation ofcellular metabolism, can lead to a much better integratedunderstanding of EPS interaction, regulation and controlwithin biofilms.

Is it all about the surface? A combination of colloidsand biology

In its simplest form, a biofilm is a consortium of cells attachedto a substrate. The requirement of a substrate for biofilmformation has therefore attracted the attention of researchersfrom polymer and surface sciences to investigate theproperties of the substrates that may encourage or discouragebiofilm formation. The high surface area‐to‐volume ratio ofbacterial cells, similar to colloidal particles, implies that theDerjaguin, Landau, Verwey and Overbeek (DLVO) theory,which dictates the adhesion of colloidal particles to surfaces,can be applied to model bacterial adhesion to substrata (Bos etal. 1999). The DLVO theory interprets particle adhesion tosurfaces as the consequence of long-range forces likeelectrostatic interactions and Lifshitz–van der Waal’s forcesbetween the particle and the surface (Poortinga et al. 2002).The analogy between bacteria and colloids highlights theimportance of characterising the bacterial cell surface, aswell as the substrate, in describing the adhesion process.Therefore, using a wealth of techniques, largely from colloid

Fig. 2 Common techniquesfrom various disciplines usedin analysing the extracellularpolymers within biofilms

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or surface sciences, several research groups have investigatedthe properties of the bacterial cell surface in relation tocolloidal stability and adhesion.

According to the DLVO theory, the stability of colloidalparticles is influenced by two key properties: (1) hydrophobic-ity and (2) surface charge. Taking a colloidal science approachto investigate bacterial adhesion and biofilm formation, it istherefore not surprising to see that research in this area hasconcentrated on characterising these two properties of thebacterial cell surface.

Busscher and Weerkamp (1987) suggested that the majorrole of hydrophobicity in bacterial adhesion is its ability toremove water from in between the contacting areas, enablingthe dehydrated parts to interact directly through short-rangeinteractions (Busscher and Weerkamp 1987). Contact anglemeasurements on dried bacterial lawns (Van Loosdrecht etal. 1987a) is a popular method to determine the bacterialsurface hydrophobicity over other techniques such as themicrobial adhesion to hydrocarbons assay (Rosenberg et al.1980), microbial adhesion to solvents assay (Bellon-Fontaineet al. 1996), hydrophobic interaction chromatography(Roettger and Ladisch 1989), two-phase partitioning testsand salt aggregation experiments using ammonium sulphate(Rozgonyi et al. 1985).

Van Loosdrecht et al. (1987a) reported a linear positivecorrelation between cell surface hydrophobicity and theadhesion of cells to a hydrophobic substrate. Van der Meiet al. (1998) also used the contact angle measurement tostudy the cell surface hydrophobicity of many different typesof bacteria and concluded that surface hydrophobicity isunique to each strain and dependent on the growth phase andgrowth rate of the strains. The conclusion implies that thepotential interaction forces that govern the adhesion betweenbacterial cells are not constant and can change depending onthe biological activity. Allison et al. (1990a, b) also found aconnection between cell surface hydrophobicity and biofilmformation with a decrease in hydrophobicity resulting in therelease of cells from P. aeruginosa and E. coli biofilms.

Van Loosdrecht et al. (1987b) recognised that anaccurate prediction of bacterial adhesion can be made whenthe surface charge of bacteria is also included in theadhesion models. Bacterial cell surface charge is generallydetermined by measuring the electrophoretic mobility andinferring the zeta potential (Wilson et al. 2001; Hong andBrown 2008), which is the electrical potential of theinterface between the aqueous solution and the stationarylayer of such a fluid attached to the bacterial cell(Klodzinska et al. 2010). Like cell surface hydrophobicity,cell surface charge has been found to vary due to biologicalactivity such as varying growth phase (Eboigbodin et al.2005; Walker et al. 2005), cellular metabolism (Hong andBrown 2010) and genetic differences (Eboigbodin et al.2006) where changes in cell surface charge subsequently

dictated the ability of the cells to aggregate (Marshall 1976;Rijnaarts et al. 1995; Eboigbodin et al. 2007).

Using techniques such as contact angle and electrophoreticmobility enables the determination of the average colloidalproperties of the bacterial population. The individual biologicalcomponents of the bacterial cell surface however give rise tothese colloidal properties, which in turn can govern the type ofinteractions leading to adhesion (Marshall 1976; Rijnaarts etal. 1995; Torimura et al. 1999; Eboigbodin et al. 2006; Hongand Brown 2008; Ojeda et al. 2008). Hence, within the fieldof biofilm research, there has been a progression towardsusing analytical chemistry techniques such as infrared andRaman spectroscopy (Mukherjee et al. 2011) and, morerecently, X-ray photoelectron spectroscopy (Yala et al. 2010)to characterise the surface chemistry of bacterial cells inrelation to adhesion.

Infrared spectroscopy has been applied in the field ofmicrobiology for more than 40 years (Heber et al. 1952;Norris 1959). FTIR has been used in microbial ecologyresearch (Nichols et al. 1985) and biofilm research inparticular (e.g. Bosch et al. 2006; Ojeda et al. 2008;Mukherjee et al. 2011). As mentioned earlier, a majoradvantage of using FTIR to characterise the chemicalfunctional groups for biofilm studies is that biofilms canbe observed non-destructively, directly, online and in realtime. As biofilms represent an extremely complex system,many different signals arise from the vibration of moleculesin the extracellular polymeric substances, cell membraneand the cytoplasm, which can contribute to the overallFTIR spectra. The FTIR spectra show characteristic peaksfor biological macromolecules and a comparison betweenthe biofilm samples and their planktonic counterparts tendto show shifts in functional groups relating to carbohydratesand proteins (Schmitt and Flemming 1998; Bosch et al.2006; Ojeda et al. 2008; Mukherjee et al. 2011).

A major drawback of using FTIR is that samples have tobe dried before analysis. Raman spectroscopy also providesinformation about the chemical composition of the samplebut within a hydrated environment. It therefore has anadvantage over FTIR and consequently has been usedextensively in biological analysis (study of tissues, bio-fluids and bacterial cells) (Beier and Berger 2009; Wagneret al. 2009; Carmona et al. 1997; Choo-Smith et al. 2001).The use of Raman spectroscopy and surface-enhancedRaman spectroscopy has also enabled the analyses ofhydrated biofilm samples, including the composition ofthe EPS (Hudson and Chumanov 2009, Ivleva et al. 2009)as well as the microorganisms embedded within it(Andrews et al. 2010; Schwartz et al. 2009). Combinationsof CLSM and Raman microspectroscopy have also enabledthe in situ comparison of biofilm formation of differentspecies grown independently (e.g. P. aeruginosa wild typeand its mutant (Sandt et al. 2009)) as well as to discriminate

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between different species of bacteria grown in biofilms (e.g.between Streptococcus sanguinis and Streptococcus mutans(Beier et al. 2010)). The combination of CLSM and Ramanspectroscopy within a hydrated environment has excellentpromise for the characterisation of the surface of multispe-cies biofilms.

The chemical information about the molecular composi-tion of the bacterial cell surface can be resolved to theatomic level using X-ray photoelectron spectroscopy (XPS)(Rouxhet et al. 1994; van der Mei et al. 2000). Thepercentage composition of the various atoms, such ascarbon, oxygen, nitrogen and phosphorus, and theirbonding environments on the cell surface can be analysedusing XPS. Several studies have focussed on linking theatomic ratios of cell surface constituents to the physico-chemistry of the cell surface in bacteria and yeasts (Hamadiet al. 2008; Ahimou et al. 2007; van der Mei et al. 2000;Amory et al. 1988). Certain studies have used XPS toanalyse environmental samples (Pastuszka et al. 2005) or toanalyse the changes on the cell surface upon changing theculture conditions (Dufrêne and Rouxhet 1996; Herben etal. 1990). The studies mentioned so far have been carriedout only on planktonic bacteria but have proved invaluableto establishing a good correlation between the physico-chemical characterisation techniques and XPS, where thecells are freeze-dried before analysis. Very few studies haveattempted to link XPS analysis and cell surface physico-chemistry of planktonic cells with adhesion to surfaces(Cerca et al. 2005; Dufrene et al. 1996; Denyer et al. 1990;Mozes et al. 1989). In these studies, XPS analysis has led tothe identification of cell surface molecules potentially

involved in the initial adhesion to surfaces. Cerca et al.(2005) noticed that, in Staphylococcus epidermidis, initialadhesion did not always correlate with subsequent biofilmdevelopment and concluded that the macromolecularmediators of cell-surface attachment maybe different tothose mediating cell–cell interactions. Whilst XPS demon-strates a good potential in identifying changes in the cellsurface macromolecular composition, future studies shouldconcentrate on comparing surface-attached cells withplanktonic cells to understand the correlation betweenphysiological changes and cell surface changes duringbiofilm formation.

The application of a combined analytical chemistry andcolloidal approach to describe the surface characteristics ofbacteria has helped to encourage multidisciplinary researchactivities (Fig. 3). However, the link between the intracel-lular activities (such as gene expression regulation) influ-encing specific biological molecules on the cell surface andthe observed changes in chemical functional groups leadingto adhesion needs further investigation. This is receivingmore attention in terms of surface proteins (see nextsection), but coupling the specific fingerprint of the cellsurface, via specific chemical analysis, with the biologicalmechanisms that result in the production of macromole-cules that are responsible for such a fingerprint is stilllacking.

In addition, recently, Mukherjee et al. (2011) andKarunakaran and Biggs (2011) have shown that there aredistinct differences in the cell surface characteristics of theadhered versus the free-floating cells. Therefore, furthercolloidal characterisation of bacteria should take into

Fig. 3 Common techniquesfrom various disciplines usedin analysing the bacterialcell surface

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account these changes when inferring or predicting inter-actions that may be responsible for biofilm formation andmaintenance.

What about intracellular activities? It is a biologicalprocess after all

Led largely by microbiologists, extensive research has beencarried out in the last few decades on identifying the geneticand proteomic basis of environmental adaptation strategieslike biofilm formation. It is now widely accepted that thephysiology of the cells within a biofilm is markedly differentfrom that of cells in the free-floating planktonic mode ofgrowth. The availability of whole genome sequencing and oftechniques such as genome profiling via microarrays hasenabled the field of biofilm research to advance beyond moretraditional microscopy-based microbiology to a more detailedanalysis of cellular metabolism.

DNA microarray technology has been used to identify theglobal gene expression profile of E. coli biofilm with severalglobal gene expression studies that are now available (see areview by Wood 2009). The DNA microarray studies of P.aeruginosa biofilms have also shown that a small number ofgenes were differentially expressed (Stoodley et al. 2002;Whiteley et al. 2001). Genes encoding for adhesion andauto-aggregation, as well as several novel gene clusters, tendto be highly expressed in biofilm cells or induced upontransition to biofilm growth (Schembri et al. 2003). Inparallel to DNA microarray assays, a transcriptome profilingtechnique has also been used to study biofilm formation.This technique has been used to study the physiology,phenotype, motility, metabolic activity and cell-to-cellcommunication in biofilm formation using some purecultures such as S. aureus, E. coli, B. subtilis, X. fastidiosa,P. aeruginosa and V. cholerae (Beenken et al. 2004; Beloinet al. 2004; Ren et al. 2004; De Souza et al. 2004; Whiteleyet al. 2001; Moorthy and Watnick 2005). However, nospecific global pattern for biofilm formation has emerged.

Nonetheless, a confirmation of the role of specific genesidentified in the global gene expression profiling is possibleusing targeted genetic mutation studies. Genetic manipulationis therefore critical for understanding and identifying charac-teristics associated with biofilm formation and adhesion(Stoodley et al. 2002; Hall-Stoodley et al. 2004; Beloin andGhigo 2005). For example, O’Toole et al. (2000) studied thebiofilm development using the crc mutant of P. aeruginosawhere the disruption of csrA gene (which is a pleiotropicregulatory protein for carbon source metabolism) increasedbiofilm formation. Whilst it is not the aim of this review todetail all genomic mutations that have led to enhanced orreduced biofilm formation, some further examples are givenin the following paragraphs.

Prigent-Combaret et al. (2000) found that the two-component regulatory systems OmpR/EnvZ (DNA-bindingresponse regulator in two-component regulatory systemwith EnvZ) and CpxR/CpxA (DNA-binding responseregulator in two-component regulatory system with CpxA)via positive and negative regulation of curli transcriptionaffect E. coli biofilm formation (Stoodley et al. 2002). Hha(osmotic stress regulator) and YbaJ (hypothetical protein)in E. coli have been shown by Barrios et al. (2006) toregulate biofilm formation as its deletion reduces thebiofilm mass. The deletion of Hha and YbaJ restoredmotility and reduced cell aggregation to that of the wild-type strain. Studies carried out by Domka et al. (2006) havealso shown that the deletion of yceP (hypothetical protein)and yliH (repressor of biofilm formation by indole transportregulation) in E. coli increases biofilm formation.

Shelud’ko et al. (2008) studied the effect of mutations inthe synthesis of lipopolysaccharides (LPS) and calcofluor-binding polysaccharides (CBPS) on biofilm formation byAzospirillum brasilense. The biofilm thickness and antigenicproperties of biofilm produced by A. brasilense Sp245 andits mutants deficient in the synthesis of LPS and CBPSshowed that mutants deficient in acidic LpsI synthesisproduced thicker biofilms on hydrophilic surfaces, and thebiofilms produced on hydrophobic surfaces by bacteria thatare unable to synthesise CBPS are less pronounced. Thestudy concluded that a fundamental change in the LPSstructure correlates with the activation of biofilm formationby the relevant mutants on hydrophilic and hydrophobicsurfaces. It also provides a useful link between surfacemacromolecules such as LPS and adhesion and could be auseful candidate to combine with the colloidal studiesmentioned in the previous section to link biological activitywith surface properties.

Specific genetic mutations in the quorum sensing system ofbacteria have also led to changes in biofilm formation. Reeser etal. (2007), when characterising Campylobacter jejuni biofilms,found that quorum sensing is required for maximal biofilmformation as the C. jejuni luxS mutant was significantlyreduced in their ability to form biofilms compared to the wild-type strain. Studies by Huang et al. (2009) have also reportedthat a luxS-dependent quorum sensing system is involved inthe formation of S. mutans biofilms. In S. mutans, knockout ofthe luxS gene was also shown to impair biofilm growth, asobserved by scanning electron, light and fluorescencemicroscopy (Sztajer et al. 2008). The deletion of transportprotein YdgG decreases the extracellular and increases theintracellular concentration of the quorum sensing moleculeauto inducer 2 (AI‐2). As YdgG enhances the transport ofAI‐2, deletion of YdgG resulted in an increase in biofilmthickness and biomass in flow cells (Herzberg et al. 2006).

In general, bacteria communicate with one another usingchemical signal molecules or auto inducers (AI). Prokaryotic

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microorganisms such as Gram-negative bacteria produce acylhomoserine lactone (AHL) as signal molecules and Grampositive-bacteria produce oligopeptides as signal molecules(Miller and Bassler 2001). Bacteria can respond to a widevariety of AI produced by the same species as well as byother genera, providing a basis for inter and intra-speciescommunication. The autoinducer-2 signal (AI-2) producedby the LuxS protein mediates interspecies communicationamong Gram-positive and Gram-negative bacteria. The roleof quorum sensing, as a genetic response, has long beenlinked to biofilm formation via both AHL and AI‐2 systems(Costerton et al. 1999; Bassler 2002; Simoes et al. 2007). Aswell as AI‐2 controlled biofilm formation as discussedabove, AHL-mediated gene expression has been shown toregulate biofilm formation in species such as P. aeruginosa(De Kievit et al. 2001), Burkholderia sp. (Brackman et al.2009) and in natural environmental samples such assubmerged limestone and water reclamation system (McLeanet al. 1997; Hu et al. 2003)

A genetic analysis provides information on the potentialof bacteria to adapt to environmental changes and biofilmformation, and mRNA measurements (via DNA micro-arrays) reveal which genes have been transcribed during theprocess. The analysis of the protein complement, on theother hand, provides direct information on the metabolicactivity occurring at the exact moment that the sample wastaken. Proteomics enables the simultaneous analysis of totalgene expression at the protein level and represents a keystrategy for studying biological systems (Hatzimanikatisand Lee 1999). Therefore, proteomics is a powerful tool forstudying the observed physiological changes occurringduring biofilm formation.

Proteomic studies using the semi-quantitative two-dimensional gel-based electrophoresis have compared theintracellular protein profiles of biofilm and free-floatingbacteria and showed clear differences in several proteinsinvolved in metabolism, transport and regulation (Otto et al.2001; Sauer and Camper 2001; Steyn et al. 2001;Oosthuizen et al. 2002; Trémoulet et al. 2002; Vilain etal. 2004a, b; Welin et al. 2003; Resch et al. 2005; van Alenet al. 2010). Advancements in proteomic analysis have alsoled to the introduction of non-gel-based quantitativetechniques such as isobaric tag for relative and absolutequantitation (iTRAQ). Mukherjee et al. (2011) and Pham etal. (2010) both used iTRAQ to quantify the intracellularprotein abundance between biofilm and planktonic cells ofE. coli and the periodontal pathogen Tannerella forsythia,respectively. A further analysis of quantitative proteomics ispossible using a variety of statistical techniques to linkprotein expression with potential metabolic pathwaysduring biofilm formation (Mukherjee et al. 2011).

Other than the soluble cytoplasmic proteins, a proteomicinvestigation by two-dimensional gel electrophoresis of the

outer membrane proteins has also been carried out, forexample, the type-1 fimbriated E. coli which revealed adistinct change in the composition of outer membraneproteins during adhesion to abiotic surfaces (Resch et al.2005; Otto et al. 2001). Pham et al. (2010), usingquantitative non-gel-based proteomic techniques, alsofound an increased expression of outer membrane proteinsin the biofilm cells.

Fig. 4 Common techniques from various disciplines used inanalysing the intracellular activities of cells in a biofilm

Fig. 5 Biofilmology, “the study of biofilms”, using a more holisticunderstanding of biofilm formation and maintenance

1876 Appl Microbiol Biotechnol (2011) 90:1869–1881

Global genomic and proteomic studies have beenextremely useful to survey the gene or protein expressionof planktonic and biofilm phenotypes and also theconsequence of isogenic mutant strains on biofilm forma-tion (Fig. 4). However, despite the wealth of research in thisarea, the studies are generally limited to sequenced micro-organisms, in particular, P. aeruginosa and E. coli, and asyet no one unique set (or cluster) of genes is responsible forthe phenotypic change during biofilm formation. Thistherefore makes it difficult to develop control strategiesbased solely on gene expression.

Research has been carried out to look at the biologicalchanges that occur as bacteria form a biofilm from aplanktonic state. However, more effort is needed to uncoverthe influence of intracellular biological changes and theireffect on the physicochemical characteristics of the bacterialsurface, which subsequently results in biofilm formation. Acomprehensive proteomic analysis of all aspects of biofilmscould provide insight into the link between biologicalprocesses (intracellular proteins), specific functional groupson the surface (surface and/or membrane proteins) andsurrounding extracellular polymers (proteins in EPS), but themajority of proteomic studies conducted until now havefocussed on planktonic rather than biofilm cells directly oronly part of the biofilm picture.

Concluding remarks

The immense potential of biofilm technologies in industry, theubiquity of biofilms in infection and disease and thecomplexity involved in studying biofilms have inspired a vastinterest amongst the research community. In order to reducethe complexities involved in studying biofilms, the research sofar has taken a parts-based approach and in so doing hasgathered vast amounts of knowledge in the various aspects ofbiofilm formation such as the role of exopolymers, theinfluence of environmental conditions and substrate propertiesand the molecular controls in biofilm formation. The creativeuse of techniques from various disciplines to examine thephenomenon of biofilm formation has brought to the forefrontthe immense potential of multidisciplinary research in com-prehending the multidimensional challenge of understandingbiofilms. Although the incorporation of multidisciplinarity inthe parts-based approach to understanding biofilms has spurredresearch into potential biofilm control strategies, the ultimateaim of specifically designing biofilms to suit the necessaryapplication is not yet achievable. In order to achieve controlover biofilm formation, researchers need to recognise that thestudy of biofilms, i.e. biofilmology, has evolved into adiscipline in its own right (Fig. 5) and that mutual cooperationbetween the various disciplines and a multidisciplinaryresearch vision are vital.

Acknowledgments The authors wish to acknowledge the UKEngineering and Physical Sciences Research Council (EPSRC) for astudentship for Karunakaran, Advanced Research Fellowship forBiggs (EP/E053556/01) and further project funding (EP/E053556/01and EP/E036252/1) and The University of Sheffield for a feescholarship for Karunakaran and Ramalingam. The authors declarethat they have no conflicts of interest.

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