Listeria monocytogenes is ubiquitous in nature and a major concern for the food industry, since it is the causal agent of the serious foodborne illness listeriosis. This organism can be introduced through many routes to food-processing environments and may become established on food-processing equipment. Subsequently, food products may become contaminated during processing. In addition, the bacterium can grow at refrigeration temperatures. Biofilms are regarded as important with respect to the survival and growth of microorganisms in the food industry. Microorganisms growing in biofilms are protected against cleaning and disinfection and are difficult to eradicate. Listeria monocytogenes may grow in biofilms that protect them against environmental stress and can be isolated from surfaces after cleaning and disinfection. For each individual food-processing plant, a limited number of clones of L. monocytogenes may become established and persist for years. Persistent strains adhere to surfaces and form biofilms more readily compared to sporadically found strains, suggesting that adherence to surfaces is important for survival and persistence of L. monocytogenes in food-processing environments. Listeria monocytogenes can adhere to all the materials commonly used in the food industry. In biofilms L. monocytogenes is significantly more resistant to disinfection than its free-living counterparts and thick, complex biofilms are more difficult to remove than adherent single cells of L. monocytogenes. Several novel approaches to avoid adhesion of L. monocytogenes have been proposed, but high costs, practical difficulties or resistance problems limit their practical use. Despite considerable research on the adhesive properties and resistance of L. monocytogenes enabling its survival in the food production environment, a final solution for avoiding establishment of the bacterium has not yet been found.

In nature, bacteria are able to form complex surface-attached communities called biofilms. Microbial biofilms pose a particular problem in many human infections because of an inherent tolerance to antimicrobial agents and host immune killing and clearance. We have used complementary DNA (cDNA) microarray technology to identify Pseudomonas aeruginosa genes that are differentially expressed in growing and developing biofilms. Our study shows that, when compared with planktonic bacteria, gene expression profiles of biofilm cells have the highest resemblance to the profiles of stationary-phase cells. We suggest that the process of biofilm development involves a series of adaptive responses including those to anaerobic and iron-limitation stresses, rather than being associated with a unique biofilm developmental program. Mapping of quorum-sensing regulated genes in a P. aeruginosa biofilm identified a set of N-acyl homoserine lactone (AHL)-dependent genes that are exclusively expressed in sessile cells. One of these genes, pvdQ, encodes an AHL acylase that degrades long-acyl but not short-acyl AHLs. This result may provide an explanation for the previous finding that the level of long-acyl AHLs is greatly reduced in P. aeruginosa biofilm cells as compared with their planktonic counterparts. Furthermore, we present evidence that quorum sensing is participating in the control of iron-limitation responses in the biofilm cells.

Here we describe an experimental study of the mechanical properties of bacterial biofilms formed from the early dental plaque colonizer Streptococcus mutans. The S. mutans biofilms demonstrated the behavior of rheological fluids, with properties similar to those of organic polymers and other biological fluids. The time-dependent response of the biofilms was modeled on the basis of principles of viscoelasticity theory. The static and dynamic responses were defined in terms of the creep compliance, storage and loss moduli, and viscosity. The creep compliance and stress relaxation functions of S. mutans biofilms were characterized using the Burger model. Implications for developing more effective mechanical removal strategies of dental plaque biofilms are discussed.

Biofilms are often considered as localized zones of high cell density. Quorum sensing provides a means for control of population processes and has been implicated in the regulation of biofilm activities. We present a role for quorum sensing in programmed detachment and dispersal processes. Biofilms of Pseudomonas aeruginosa PAO1 and its isogenic homoserine lactone (HSL) mutant P. aeruginosa PAO-JP2 were grown in batch culture on glass substrata; differences were found in the rate and extent of formation of biofilm. Climax communities were observed for PAO1 at 24 h. These were later accompanied by foaming, a drop in the surface tension of culture media and dispersal of the biofilm, after which no subsequent biofilm accretion occurred. PAO-JP2 cultures reformed biofilm post-detachment and did not foam. Prevention of biofilm reformation in the wild type was related to some component excreted into the culture medium. Rhamnolipid, a biosurfactant regulated by quorum sensing, was detected in PAO1 cultures. When rhamnolipid was added to freshly inoculated substrata, biofilm formation was inhibited. At 20 h, PAO1 biofilms were transferred to medium with added rhamnolipid: biofilm was relatively unaffected. Biofilm events were also studied in medium supplemented with N-butyryl-L-homoserine lactone, which is involved in the regulation of rhamnolipid synthesis. Both strains exhibited similar trends of rapid biofilm formation and dramatic changes in the rate and extent of biofilm accretion. In both cases, there was premature foaming, lowered surface tension and elevated rhamnolipid levels. A role for HSLs in maintenance of biofilm and events leading to dispersion of cells is proposed. This role would encompass dispersion but not necessarily detachment of cells from biofilm and supports a new function for rhamnolipid in pathogenesis, whereby rhamnolipid would promote the dissemination of cells from a nidus of infection.

Biofilms colonizing ancient limestone Mayan monuments in Uxmal (Yucatán, Mexico) were characterized for their microbial composition and differences using phospholipid fatty acid (PLFA) analysis and single-strand conformation polymorphism (SSCP) of polymerase chain reaction (PCR)-amplified genes of the rRNA small subunit. Biofilms 1–7, displaying different macroscopic characteristics, were drawn from interior and exterior limestone walls, which were characterized by different microenvironmental conditions. Inner surfaces that were exposed to low light intensity and higher water availability supported a higher biofilm biomass, probably indicating that these environmental conditions are more suitable for biofilm growth and development. Sequencing of SSCP profiles from the biofilms showed that bacteria affiliated with the phyla Proteobacteria, Bacteroidetes and Cyanobacteria colonized both internal and external surfaces, although certain Nevskia and Salinibacter were particularly associated with internal biofilms. In contrast, a particular biofilm community composed almost exclusively of Rubrobacter-related bacteria was found in only one exterior partially shaded sample (biofilm 3), characterized by high illumination and low water content. No specific organisms were detected in biofilm 7. The PLFA profiles suggested that cyanobacteria were the biomass-dominant group in most biofilms, except for biofilm 3 (exterior) where actinobacterial markers were detected in significant proportions. Interestingly, most of the detected sequences were related to halophylic bacteria, although the similarity of these clones to known sequences in databases was low (< 94%). This finding suggests that much bacterial novelty, probably of halophylic nature, remains to be identified in these biofilms. Our study suggests that water availability and light regime appear to be the main environmental determinants defining boundaries for biofilm formation, while substratum salinity appears to be an important abiotic factor that influences biofilm community structure in these specialized microbial habitats.

The desmid Penium margaritaceum is a common resident of biofilms of shallow Adirondack wetlands in New York State, USA. It was isolated and grown in the laboratory where it readily formed biofilms and produced large amounts of extracellular polymeric substance (EPS). The EPS was separated into two fractions: an EPS gel and soluble EPS. Both fractions were rich in xylose, fucose and glucuronic acid. The EPS gels contained large amounts of 3-linked, 4-linked and 3,4-linked fucose, 3,4-linked glucuronic acid and terminal xylose linkages. The EPS gel consisted of a fibrillar matrix that linked cells and cell substrate together. Immunofluorescence analysis using an anti-EPS antibody revealed that EPS secretion occurs in several different modes, which contributes to initial adhesion, capsule formation and gliding.

We present experimental results of colony formation in bacteria as an example of pattern formation resulting from reproduction and movement in biological populations. The bacterium Bacillus subtilis is known to exhibit at least five distinct types of colony pattern, depending on the substrate softness and nutrient concentration: diffusion-limited aggregation (DLA), compact Eden-like, dense branching morphology (DBM), concentric ring-like, and disk. We established a morphological diagram of the colony patterns, and then examined and characterized both macroscopically and microscopically how the the colonies grow. There seem to be two kinds of bacterial cells – active and inactive – and the active form drives the colony interfaces outwards. The active cells may be clearly distinguished from the inactive ones as they form the characteristic fingernail-like structure at the tips of growing branches of the DBM colony. The concentric ring-like colony is formed as a consequence of repeated alternate migration and resting of the growing interface, the cycle time for which seems to be independent of the substrate softness and nutrient concentration. So far there have been several phenomenological models proposed to qualitatively explain or reproduce the patterns observed in bacterial colonies. A few of them are reviewed here, systematically and critically, in light of our experimental results.

Microbial biofilm formation can be influenced by many physiological and genetic factors. The conventional microtiter plate assay provides useful but limited information about biofilm formation. With the fast expansion of the biofilm research field, there are urgent needs for more informative techniques to quantify the major parameters of a biofilm, such as adhesive strength and total biomass. It would be even more ideal if these measurements could be conducted in a real-time, non-invasive manner. In this study, we used quartz crystal microbalance (QCM) and microjet impingement (MJI) to measure total biomass and adhesive strength, respectively, of S. mutans biofilms formed under different sucrose concentrations. In conjunction with confocal laser scanning microscopy (CLSM) and the COMSTAT software, we show that sucrose concentration affects the biofilm strength, total biomass, and architecture in both qualitative and quantitative manners. Our data correlate well with previous observations about the effect of sucrose on the adherence of S. mutans to the tooth surface, and demonstrate that QCM is a useful tool for studying the kinetics of biofilm formation in real time and that MJI is a sensitive, easy-to-use device to measure the adhesive strength of a biofilm.

Biofilms in drinking-water distribution systems may accumulate human pathogenic viruses. Viruses that attach to biofilm are removed from the water phase improving the water quality. However, if released in slough, it may still present a risk of infection. This review describes the available data on the presence of pathogenic viruses in drinking-water biofilms. First, biofilms of distribution systems potentially contribute to viral contamination of tap water only if infectious viruses are present initially in the water, which has been shown in several recent studies. However, only one out of three field studies showed the presence of infectious enteroviruses in natural biofilms from drinking-water networks. The presence of pathogenic viruses in biofilms points to the ability of these viruses to attach to biofilms. This has also been shown in pilot-scale studies in which bacteriophages and vaccine poliovirus strains were spiked into water and could be eluted from artificial biofilms. Reported attachment rates vary greatly and may depend on many factors such as the biofilm characteristics, the virus strain and the efficiency of viral recovery from biofilms. One study reported biphasic viral inactivation in biofilms with rapid initial inactivation followed by slow inactivation, implying that some of the attached viruses are able to remain infectious for a longer time. In several laboratory experiments, virus attachment to biofilms has been reported under various conditions; however, although detachment of sloughs in distribution systems has been observed, the presence of viruses in these sloughs was not studied. Here, we discuss the possible presence of infectious pathogenic viruses in sloughs; the extent to which these will pose a health risk remains to be investigated.

Biofouling in water distribution systems has, arguably, affected our lives for more than 3500 years. It may be defined as the undesirable accumulation of biotic matter on a surface, which can cause odour and taste problems, the deterioration of pipe materials and fittings and result in the discoloration of water. Early efforts to combat these problems included the use of sedimentation tanks, disinfection by silver ionization and cleaning of the distribution network. At the turn of the nineteenth century, rapid sand filtration and water disinfection became widely used and helped to reduce the organic and bacterial load in drinking water. A better understanding of the role and causes of biofouling in water distribution systems resulted in various legislations, which in turn have been a driving factor for improving or developing new water treatment methods, pipe materials, analytical techniques, etc. However, increasing requirements on water quality in the late twentieth century made water treatment and specific anti-corrosion and/or microbial control regimens insufficient as a means of solving the transportation problem owing to the heterogeneity of pipe materials and contamination from outside the distribution system. Furthermore, as drinking water passes through the mains it undergoes a series of quality changes owing to interactions with the pipe walls, bacteria and the sediment phase.

This review emphasizes the extent to which biofouling depends on interactions between microorganisms and (1) nutrients, (2) environmental conditions (temperature), (3) physicochemical processes (sedimentation, corrosion, disinfection) and (4) pipe material. A good knowledge of these complex interactions is necessary for implementing a successful biofouling control strategy.

The study of bacterial colonies is a crucial step towards understanding biofilms. We review some of the exciting discoveries about bacterial self-organization that might shed new light on biocomplexity in general and biofilms in particular. This review is aimed at researchers from different disciplines – microbiology, biology, chemistry, physics, mathematics and computer science. To make the presentation comprehensible we have avoided the use of specialized terminology of the different disciplines and limited the experimental and computational details.

Bacteria can self-organize into hierarchically structured colonies of 109 to 1012 bacteria, each utilizing a great variety of biochemical communication agents, such as simple molecules, polymers, peptides, complex proteins, genetic material and also “cassettes of genetic information” such as plasmids and viruses. Bacteria use their intracellular flexibility, involving signal transduction networks and genomic plasticity, to collectively maintain self and shared interpretations of chemical cues, exchange of meaning-bearing chemical messages, and dialogues. The meaning-based communication permits the formation of colonial intentional behavior, purposeful alteration of colony structure and decision-making – features we might begin to associate with bacterial social intelligence. Such social intelligence, should it exist, would require going beyond communication to encompass additional intracellular processes, as yet unknown, for generating inheritable colonial memory and commonly shared genomic context.

Myxococcus xanthus is a motile soil bacterium with complex social behaviors. Upon starvation, a developmental program is initiated that results in cellular aggregation and fruiting body formation. This process requires the exopolysaccharide (EPS) component of the extracellular matrix. With prolonged starvation, a part of the cells within a fruiting body die, while the other cells differentiate into spores. Extensive genetic and biochemical information has been generated that elucidates this interesting developmental process. Little is known, however, about the detailed three-dimensional structural features of native fruiting bodies or the EPS and distribution of live/dead cells (spores) within these structures. In this study, changes in the three-dimensional architecture of fruiting bodies and the distribution of the extracellular matrix within the fruiting bodies during the developmental process were investigated using a gfp-expressing M. xanthus strain and carbohydrate-specific lectins or monoclonal antibodies in combination with confocal laser scanning microscopy. The extracellular matrix was found to form a scaffold within the fruiting body structure. Furthermore, using a bacterial viability staining assay, the distribution of live/dead cells within fruiting bodies was examined at different times. The majority of live cells were found to localize at the outer layer of a mature fruiting body, with dead cells underneath.

Mathematical modeling of spatial biofilm structure has been in development for the past 10 years, its main goal being to derive the dynamics of biofilm structure from first-principle descriptions of the various physical, chemical and biological processes involved in biofilm formation. Early efforts described development of unrestricted monospecies consortia, often considering diffusion and reaction of a single solute species. Multi-dimensional modeling of biofilms has presently reached a stage where multi-species systems with any number of bacterial and solute species, reactions and arbitrary detachment scenarios may be readily implemented using a general-purpose software framework introduced recently. The present work presents motivations for the mathematical modeling of biofilm structure and provides an overview on major contributions to this field from pioneering efforts using cellular automata (CA) to more recent methods using the preferred individual-based modeling (IbM). Recent examples illustrate how biofilm models can be used to study the microbial ecology in: (a) development of multi-species nitrifying biofilms with anammox bacteria, (b) interspecies hydrogen transfer in anaerobic digestion methanogenic consortia, (c) competition between flock-formers and filamentous bacteria influenced by environmental conditions and its effect on morphology of activated sludge flocs, and (d) a two-species biofilm system with structured biomass describing extracellular polymeric substances (EPS) and internal storage compounds. As recent efforts from direct comparison of structure predicted by three-dimensional modeling with that observed by confocal laser scanning microscopy imaging of biofilms grown in laboratory flow cells show a good agreement of predicted structures, multi-dimensional modeling approaches presently constitute a mature and established methodology to enhance our understanding of biofilm systems.

Two ultrasonic devices – flat (T1) and curved (T2) ultrasonic transducers – were developed to remove biofilms from opened and closed surfaces, respectively. The aim is to standardize biofilm removal for in situ sanitary control in the food industry. The biofilms studied in this work were model biofilms made with milk on stainless steel sheets. We have shown in a previous study that sonication could be employed to remove and resuspend biofilm consistently, with a good recovery rate, from opened surfaces. Plate counting was used to assess the efficiency of each treatment. A total removal of Escherichia coli and Staphylococcus aureus from model biofilms was obtained with T1: 10 s at 40 kHz. However, ultrasound applied with T2 (a patented curved transducer developed for closed surfaces: 10 s at 40 kHz) failed to completely remove these model biofilms: 30±7% and 66±10% for E. coli and S. aureus biofilms, respectively. In order to improve the biofilm removal from closed surfaces with T2, the effect of the application of ultrasound in combination with chelating agent preparations was investigated. The application of ultrasound with T2 in 0.05 mol EDTA or EGTA per litre dislodged the E. coli milk model biofilm, with 100±10% and 100±5% recovery yields, respectively. These results showed a synergism between ultrasonic waves and chelator preparations, i.e. the combination achieved three times the recovery rate of sonication alone (30%). However, when the same treatment was applied to the S. aureus milk model biofilm, the combined treatment with EDTA or EGTA did not significantly improve the recovery of the biofilm cells: 74±26% with EDTA at 0.025 mol/l and 41–47% with EGTA at 0.025 mol/l and 0.05 mol/l, respectively, compared with 66±10% for sonication alone. The combined treatment was in agreement with an industrial control, i.e. a good reproducible recovery of the biofilm in a few seconds (10 s) for E. coli milk biofilms but not for S. aureus biofilms.

Bacteria in nature grow mostly as biofilms, surface-associated cells enveloped by hydrated extracellular polymeric substances (EPS) of bacterial origin. The composite of EPS and biofilm cells is measured when quantifying bacterial biomass from natural samples. However, little is known regarding the relative magnitude of the EPS and cellular fractions of biofilm biomass, particularly in unsaturated systems such as soil and food surfaces. In this study, we examined the cellular and extracellular fractions of Pseudomonas aeruginosa biofilms for DNA, protein and carbohydrate content. Biofilms were cultured in a model laboratory system that simulates the nutrient gradients and poorly mixed nature of unsaturated systems. We found that unsaturated biofilms exhibited two growth phases – an initial rapid phase and a second phase of slower or negligible development. However, no lag phase was observed for either carbon source. Cellular DNA accumulated linearly with biomass whereas cellular protein and carbohydrates accumulated exponentially with biomass. High levels of carbohydrate, protein and DNA were observed in the EPS of all samples, representing as much as 50% of these macromolecules in the biofilm. Most EPS accumulated during the second phase of growth, when cellular DNA increased only slightly. However, for biofilms cultured on a poorly bioavailable carbon source, EPS DNA decreased during the second phase, suggesting that EPS may affect bacterial survival under nutrient-limited conditions. Whether a product of overflow metabolism or cell lysis, EPS is a significant component of unsaturated biofilm biomass that probably impacts on bacterial ecology.

A rheological study was performed on biofilms of mucoid Pseudomonas aeruginosa strains producing high molecular weight alginate as the major polysaccharide component of the extracellular polymeric substances (EPS). Environmental strain SG81 and clinical strain FRD1 produced O-acetylated alginate, and mutant FRD1153 derived from strain FRD1 was defective in alginate acetylation. Confluent biofilms were prepared by cultivating the bacteria at 36 °C for 24 h on membrane filters placed on Pseudomonas isolation agar. The rheological properties of these pure culture biofilms attached to the membrane filters were analysed using a rotating disc rheometer. The biofilms of all mucoid strains showed viscoelastic properties, with polymer (alginate) cross-linking caused predominantly by physical interactions in the form of entanglements. In dynamic frequency-sweep tests, we observed time-dependent plateau regimens, describing the dynamics of network structures. From these data, we could derive the concentration of elastically effective polymer chains as 5 (±1) x 1019 chains/l (limits are standard deviation) for 24 h old biofilms of P. aeruginosa SG81. Relaxation processes predicted an average lifetime of junction points of the order of 16 (±10) s. Pseudomona aeruginosa FRD1 revealed a more elastic polymer network when compared with the acetylation-defective strain FRD1153, which had a more viscous biofilm structure. With calcium (10 mmol/l) added to the growth medium, enhanced biofilm stability was observed, which was based mainly on Coulomb interactions between divalent calcium ions and the carboxylate groups of alginate, with entanglements making only minor contributions. The calcium-induced increase in polymer network stability was greater in biofilms of P. aeruginosa FRD1 than in the acetylation-defective strain FRD1153. These results indicate that acetyl groups as low molecular weight substituents of alginate polymers strongly influence the rheological behaviour of mucoid P. aeruginosa biofilms and are involved in the stabilization of polymer networks of the EPS matrix in both the absence and the presence of calcium ions.

This study examined quantitative (dry weight, chlorophyll a content and C : N ratio) and qualitative (community compositions of bacteria and diatoms) changes in marine biofilms as a function of season (summer 2003 and winter 2004), temperature (16, 23 and 30°C) and salinity (20‰, 27‰ and 34‰) under laboratory conditions. Biofilms were allowed to develop for 20 days in the laboratory, using natural sea water collected from Port Shelter, Hong Kong. The following results were obtained: (1) biofilm dry weight was greater in summer than in winter, and greater at 34‰ than at 20‰; (2) biofilm chlorophyll a content was affected by all three factors (season, temperature and salinity), with significant interactive effects among the three factors; and (3) C : N ratio was affected by season (winter > summer) and temperature (30°C>16°C in summer), but not by salinity. Bacterial community composition was analyzed by terminal restriction-fragment length polymorphism of polymerase chain reaction-amplified 16 S rRNA genes. In summer, community compositions of both bacteria and diatoms were strongly affected by salinity. In addition, natural summer biofilms that developed at three field sites, where different salinities were found, harbored appreciably different bacterial and diatom community compositions. In contrast, in winter, temperature exerted a major influence on community compositions. The present study adds to the growing evidence that environmental factors are important determinants of both the quality and quantity of marine biofilms.

Planktonic larvae of many sessile marine invertebrates settle and metamorphose preferentially on surfaces covered by bacterial biofilms. The polychaete tubeworm Hydroides elegans is induced to settle by biofilms and is the primary colonizer of newly submerged surfaces in the succession of macrofouling invertebrates in Pearl Harbor, Hawaii, USA. This study examines the succession of bacterial community composition and cell density of marine biofilms, and how temporal changes in biofilms affect settlement of H. elegans. Settlement assays of H. elegans were conducted on naturally formed biofilms of increasing age from Pearl Harbor. Denaturing gradient gel electrophoresis and epifluoresence microscopy were used to identify community composition and densities of bacterial cells in biofilms. Results of this study suggest that increasing densities of a stable community, rather than a shift in dominant species composition of biofilm bacteria, are probably responsible for the primary colonization of submerged surfaces by H. elegans in Pearl Harbor. However, the current data leave open the question of why larvae of other biofouling invertebrate species known to settle in response to biofilms recruit to surfaces only at later times.

High cell density and close proximity of diverse species of microorganisms are typical of life in natural biofilms. These conditions give ample opportunity for both competitive and cooperative interactions between individuals of the same and different species. Cooperative behaviour benefits the group of neighbouring microbes but comes at a fitness cost for the cooperating individuals. This creates a conflict of interest between the fitness of the individual and the fitness of the group. Individuals that defect from cooperation and therefore do not pay the cost but nevertheless benefit from the cooperative behaviour of others are called cheaters. Cooperative behaviour in the presence of cheaters constitutes altruism towards the cheaters. The aim of this review is two-fold: first, to introduce key concepts from kin selection and group selection theory that allow us to understand how cooperative behaviour can evolve in the face of cheaters; secondly, to draw attention to the conflicts of interest prevalent in biofilms yet largely ignored in the biofilm literature. Examples discussed comprise growth restraint in stationary phase as an instance of the Prisoner's Dilemma, growth restraint to allow channel formation, restraint in resource consumption or economical use of resources as altruistic behaviour, population heterogeneity as insurance against environmental changes, cooperative investment in diffusible exoenzymes, cooperation of pathogens and virulence, diffusion sensing versus quorum sensing and the inflation of signals, antibiotic resistance as collective action, and programmed cell death.

In spite of the large range of morphologies observed for biofilms, there is strong experimental and theoretical evidence that the complex nature of biofilm structure dynamics is primarily a consequence of the effect of environmental conditions on biofilm development. It has been observed from the operation of industrial and laboratory-scale biofilm reactors that the structure of biofilms results from a balance of the detachment forces and the regimen of transport of a growth-limiting substrate. The overall performance of biofilm reactors is intrinsically dependent on biofilm morphology. The spatial distribution of the diverse dissolved and particulate components through the biofilm matrix and the shape of its external surface influence the rates of the occurring bioconversions, and structure also influences the stability of the biofilm in terms of resistance to mechanical stress. Individual-based modelling (IbM) of biofilms structure dynamics is used here to unify observations from the operation of biofilm reactors by simulating biofilm growth under variable detachment forces and mass transport regimens for a growth-limiting substrate. The IbM is a bottom-up approach, where the global system behaviour is derived from the local interactions of multiple elements acting independently. Transport and reaction of a solute species, local microbial growth rates and the effect of external detachment forces applied to the biofilm are modelled using differential approaches. Simulations carried out in two-dimensional space using this model illustrate a range of biofilm morphologies that emerge from different reactor operation parameters, reproducing trends observed experimentally. Comparison of multi-dimensional modelling results with those obtained using one-dimensional approaches enforces the need to use multi-dimensional modelling to predict properties that derive from the spatial biofilm structure.