Interactions between sulfur and metals were studied in aerobic and anaerobic biofilms grown on domestic waste water at 15°C. The dominant metals in the waste water were iron, zinc and copper, which were present in average concentrations of 0.5mg/l, 0.6mg/l and 0.1m/l, respectively. Copper and zinc were found to accumulate in the anaerobic biofilm owing to precipitation of metal sulfides. Iron supplementation (approximately 5.5mg Fe/l) to the anaerobic waste water reduced the zinc and copper precipitation due to sulfide precipitation with iron. However, even at these elevated iron concentrations in the waste water, sulfide precipitation in the biofilm was controlled largely by zinc and copper. In the aerobic biofilm, addition of iron resulted in accumulation of iron and phosphate, probably owing to precipitation of iron phosphates and iron (oxy)hydroxides. The potential importance of extracellular polymeric substances (EPS) on metal sorption in sewer biofilms was studied. EPS consisted mainly of proteins (13–260mg/(g volatile solids)) and to a lesser extent of carbohydrates (8–26mg/(g volatile solids)). The EPS composition remained relatively constant during experimental runs, but differed significantly between them. No relationships between the metal content of the biofilm and the amount of extracted EPS were found, which suggests that EPS did not play a major role in the metal accumulation.

The susceptibility of Pseudomonas aeruginosa (PA01) bacteria to ultraviolet-A (UV-A) light in photocatalytic and non-photocatalytic systems was investigated. Thin films of TiO2 were deposited on glass slides using a dip-coating method. Disinfection of both planktonic and biofilm bacterial cells was studied over both coated and uncoated slides exposed to low intensity (1.0 mW/cm2) UV-A irradiation. Photocatalytic treatment of planktonic PA01 cultures pro-vided a 4-log reduction in the number of viable cells in 3 h whereas UV light alone produced a 1-log reduction. For biofilm bacteria, photocatalytic treatment did not enhance killing relative to UV treatment alone; however, the UV illumination resulted in a 2- to 3-log reduction in viable cells as compared with non-illuminated controls. Possible explanations for the discrepancy between susceptibility of planktonic and biofilm bacteria to photocatalytic oxidation are discussed.

Four bacterial isolates (Chryseobacterium sp., Pseudomonas fluorescens and two Stenotrophomonas maltophilia isolates) originally isolated from the water system aboard the Mir Space Station were grown in two concentrations of nutrient broth under modeled reduced gravity using clinorotation. Sampling was performed over a 7 day period and planktonic cells were enumerated using 4′,6-diamidino-2-phenylindole (DAPI), while those attached to stainless steel were enumerated using the LIVE/DEAD® BacLight™ kit and DAPI. On some of the sampling days for all the isolates, planktonic cell counts were higher under modeled reduced gravity as compared with the normal gravity controls. In contrast, the number of cells of P. fluorescens and one S. maltophilia isolate attached to the stainless steel disks was higher under modeled reduced gravity as compared with normal gravity, whereas no such differences were observed for Chryseobacterium sp. and the other S. maltophilia isolate. Differences in motility among isolates appeared to influence the growth of planktonic cells under modeled reduced gravity but did not appear to be related to biofilm formation.

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.

Epilithic biofilms play key roles in rocky shore ecosystems. The diversity and biomass of epilithic biofilms were determined along a subtropical intertidal rocky shore at Xpicob, Southern Gulf of Mexico (Campeche, Mexico). Biofilm diversity was assessed by polymerase chain reaction (PCR) amplification of partial 16 S rRNA genes using single-strand conformation polymorphism (SSCP). Triplicate 2 cm×2 cm biofilm samples per site were taken randomly from 0.6 m×0.6 m quadrats located in two sampling sites, established 20 m apart within a homogeneous calcareous intertidal platform. Twenty-two partial rRNA sequences, belonging to four bacterial divisions (Cyanobacteria, Bacteroidetes, Actinobacteria and Proteobacteria), were recovered from these biofilms; of these, cyanobacteria were the most abundant (41%). The occurrence of cyanobacterial sequences in most samples, along with the detection of high levels of chlorophyll a and phycobiliprotein, indicates that these organisms are dominant within the biofilms. Consistent with previous reports, thick-sheathed cyanobacteria such as those found in this study (Xenococcus, Myxosarcina and Chroococcidiopsis) are typical in habitats in intertidal zones. In addition, most of the detected organisms from other bacterial lineages had closest relatives displaying biofilm phenotypes that suggested stressful conditions (i.e. desiccation) prevailed in the intertidal shores; this was selecting for biofilm-forming or thick-sheathed organisms as an ecological adaptation to withstand the conditions. Unweighted pair-group method with arithmetic mean (UPGMA) analysis of SSCP profiles indicated that there was significant spatial heterogeneity in biofilm community composition across different scales of analysis and that this heterogeneity was related to the distance at which samples were taken. This analysis showed that certain organisms appeared to be station specific and that the biofilm community structure varied even on a small scale (1 cm). Since no significant differences in bulk sea water or substratum physicochemistry were observed, these results suggest that small-scale variability of environmental conditions, surface properties and/or biological interactions (i.e. allelopathy) may be important factors structuring these biofilm communities.

This review details the current literature on relevant aspects of biofilm formation and microbial control in dental practice. To date, there is no published evidence of a serious public health risk from biofilm-contaminated dental waterlines. However, there remain few effective methods of decontamination of such waterlines.

Infrared spectra of Pseudomonas aeruginosa PAO1 biofilms grown on 40° ZnSe and 40° AMTIR (amorphous material transmitting infrared radiation, Ge33As12Se55) internal reflection elements (IRE) at 26 °C in a flow cell were collected. The depth of penetration of the evanescent wave was sufficient to identify four biofilm stages during the experiment: (1) cell/substratum attachment, (2) lag phase, (3) later-stage growth, and (4) restructuring (e.g. rearrangement, detachment or “erosion”). Our experimental results provide the first detection via a non-perturbative technique of an excess of protein in the neighbourhood of the surface. Our mathematical analysis applied to the data supports the conclusion that our observations cannot be completely accounted for by the changing characteristics of bacterial proteins but, rather, shows that an accumulation of excess protein near the attachment surface is taking place. On the assumption that biofilm cells are approximately of the same size as those of the planktonic culture, this suggests the presence of extracellular proteins. Our data do not support the presence of extracellular nucleic acids near the attachment surface in these biofilms. This conclusion is based on our observation that, during the initial 20 h of development (stage 2 and the beginning of stage 3), biofilms had a higher proportion of protein to nucleic acid than in the latter part of stage 3 and in stage 4. In the restructuring phase this ratio became equal to the ratio determined for planktonic cultures. The decrease of biomass during stage 4 is inconsistent with the physical removal or sloughing-off of cellular aggregates from biofilms within a few micrometres of the substrate surface but, instead, supports a model of gradual cell migration away from it. During this cell migration there is also a gradual loss of extracellular protein. Alternatively, therefore, the restructuring may involve cell lysis and the observed loss of biomass may be due to soluble macromolecules being removed from the neighbourhood of the surface.

The aim of this work was to study the community level physiological profile of a developing oral biofilm. Mixed-species biofilms, derived from human saliva, were grown in a constant-depth film fermentor for up to 240 h. Biolog GP2 microplates were inoculated with saliva or homogenized biofilms and incubated at 37 °C. The results showed that a biofilm community could utilize a range of substrates more diverse than those of their planktonic counterparts. Following the construction of dendrograms, a temporal relationship was demonstrated between the inoculum and biofilms, and the difference in communities over time was also resolved by using an eight-digit code of positive carbon sources. The diversity of the populations could be determined through the use of the community level physiological profile and the treatment of the data may augment the interpretation of the results. Methods such as community level physiological profiling may provide a valuable insight into the composition of biofilm communities associated with oral health and disease.

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.

Biofilm growth within a water distribution system could lead to operational problems such as pipe corrosion, water quality deterioration and other undesirable impacts in water distribution systems. With the high ambient temperatures experienced in Singapore, the operating environment in water distribution systems is expected to be more conducive to biofilm development. We have recently conducted a survey on biofilms potentially present in a local water distribution system.

The survey results indicated that residual chlorine (±standard deviation) decreased from 1.49±0.61 mg/l (water plant outlets) to 0.82±0.21 mg/l (block pipes) or 0.18±0.06 mg/l (unit pipes), respectively. Consumed chlorine, instead of residual chlorine, was found to be correlated with biofilm bacterial population. Assimilable organic carbon (AOC) level was 160±66 μg acetate C/l, and AOC:PO4-P:NO3-N was about 8:13:1. Carbon source seemed to be the limiting nutrient for bacterial growth. The concentration of iron increased from <0.04 mg/l (water plant outlets) to 0.22±0.10 mg/l (all sites). All samples showed negative results in a coliform test. The average heterotrophic plate count (HPC) for the suspended bacteria was 20 colony-forming units (c.f.u.)/ml (2 days, 35 °C) or 290 c.f.u./ml (8 days, 35 °C). The average HPC for the biofilm bacteria was 6500 c.f.u./cm2 (2 days, 35 °C) or 29000 c.f.u./cm2 (8 days, 35 °C). High HPC values in samples B2a, B2b and B3a (representing biofilm samples at site 2 from block/unit pipes and biofilm samples at site 3 from block pipes, respectively) illustrated that the relevant sample sites had a higher probaboility of biofilm growth.

In previous work, we described a protocol for measuring biofilm formation on opaque and non-opaque surfaces using reflected light. In the present work, we used the reflectance assay to assess biofilm formation for 14 strains of Enterococcus and compared our results with those obtained using a Crystal Violet (CV) assay. In general, the results for the two assays were in agreement: both identified the same Enterococcus strain as the highest biofilm-forming strain and the same two strains as the lowest biofilm-forming strains. However, two Enterococcus strains identified as high biofilm formers by the CV assay were not identified as such by the reflectance assay. A single numerical value was obtained for each of the reflectance assay results that corresponded well with the confluence of a biofilm (and presumably its depth) using phase contrast microscopy.

The effect of growth and detachment on formation of large-scale biofilm structure

Biofilm cohesive energy density determination using a novel atomic force microscopy methodology

Fluorescence correlation spectroscopy under two-photon excitation for the study of diffusion and reactivity of bacteriophage inside bacterial biofilms

Biothermodynamic characterization and dynamic analysis of biofilms using calorimetry

Biomimetic antifouling coatings for sensor surfaces for water monitoring: performance control in defined biofilm cultures and under real environmental conditions

The contribution of rpos to formation of Escherichia coli biofilms

Synergistic effects in mixed Escherichia coli biofilms: conjugative plasmid transfer drives biofilm expansion

The universal stress protein PA3309 in Pseudomonas aeruginosa is induced in biofilms

Extracellular polymeric substances from biofilms on membranes in waste-water treatment plants

Biofilm-to-planktonic cell yield: a strategy for proliferation

Physiological and phylogenetic characterization of the dispersed and loosely attached fraction of activated sludge flocs

Towards a deterministic model of biofilm detachment: an experimental study

Effect of backwash on the characteristics of biofilm in a biological activated filter reactor using elemental sulfur particles

Process performance and biomass properties in membrane-aerated bioreactors

Bioaugmentation via conjugation in biofilms treating 3-chloroaniline: effects of selective pressure

Effect of phosphorus on biofilm growth in a completely mixed biofilm reactor

Impacts of biofilm development on reactive transport in porous media under variable flow regimens

Influence of biofilms on colloid mobility in the subsurface

Biofilms in amendable in situ microcosms indicate relevant electron acceptor processes at a BTEX-contaminated aquifer

Functional biodiversity of complex biofilms grown on polychlorinated biphenyl oil

Identification and characterization of biofilm formation phenotypes of several clinically relevant Streptococcus pyogenes serotype strains

Selected probiotic bacteria disrupt biofilm development of vancomycin-resistant Enterococcus faecium

Comparison of the extracellular polymeric substances of Candida albicans and Candida dubliniensis biofilms

Influence of quorum-sensing regulated production of an antimicrobial component by Serratia plymuthica on establishment of dual species biofilms with Escherichia coli

Biofilm formation by the thermophilic and cellulolytic actinomycete Thermobifida fusca

Biomonitoring of bacterial contamination on different surfaces of food-processing machines

Role of the flagella during the adhesion of Listeria monocytogenes EGD-e to inert surfaces after cultivation at different pHs and temperatures

Adhesion of Saccharomyces cerevisiae to stainless steel: influence of surface properties

Investigating the mechanical strength of biofilms with fluid dynamic gauging

Three-dimensional biofilm model with individual cells and continuum extracellular polymeric substances matrix

A three-dimensional computer model analysis of four hypothetical biofilm detachment mechanisms

Modelling biofilm growth, detachment and fluid flow in a cross-section of tube reactors

Biofilm games

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.

We have compared the outer membrane protein (OMP) pattern of sessile Pseudomonas aeruginosa cells with that of its stationary-phase free-floating counterparts. Two-dimensional electrophoretic gels were compared using computing scanning densitometry. The level of 103 OMPs (i.e. about 50% of the spots that fulfilled our statistical selection threshold) was significantly and reproducibly different in biofilm organisms as compared with their planktonic counterparts. Some OMPs that specifically either accumulated or decreased were identified by mass spectrometry and the role of these proteins in the biofilm phenotype is discussed.

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.

Bacteria often choose a sessile biofilm lifestyle as a strategy to overcome environmental stress. In this study, we describe biofilm formation by Helicobacter pylori on a polystyrene surface, evaluating the viability and the morphological dynamics of bacterial cells during multicellular aggregate development. Moreover, we investigate expression of the luxS and rpoD genes, which are involved in biofilm formation.

Two clinically susceptible and resistant strains of H. pylori were analyzed, as well as H. pylori ATCC 43629 for reference. The dominant form of expression, clustered bacterial cells arranged in an abundant matrix, was recorded after 2 days of incubation without shaking. Coccoid (spherical) cells with a “wrinkled” aspect presented the prevalent morphology (59.26%) among cells in the biofilm structure as observed by scanning electron microscopy. In aged H. pylori cultures, death occurred in single cells or cells grouped into microcolonies in which degenerated bacteria were localized inside the aggregates. The expression of luxS and rpoD genes among the sessile bacterial population reached a maximum after 2 days, with a significant reduction at subsequent time-points. No differences in gene expression and biofilm formation were recorded in the three evaluated strains.

The morphological fickleness expressed in the life cycle by H. pylori strains emphasizes the bacterium's ability to overcome environmental stress, balancing its spread both outside and inside the host.

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 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.

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.

This list includes conferences that had been announced at the time of going to press. We are happy to include details of other biofilm-related conferences – please send details of these to the editor-in-chief.